Molecular profile of driver genes in lung adenocarcinomas of Brazilian patients who have never smoked: implications for targeted therapies.

INTRODUCTION: Lung cancer in never-smoker (LCINS) patients accounts for 20% of lung cancer cases, and its biology remains poorly understood, particularly in genetically admixed populations. We elucidated the molecular profile of driver genes in Brazilian LCINS. METHODS: The mutational and gene fusion status of 119 lung adenocarcinomas from self-reported never-smoker patients, was assessed using targeted sequencing (NGS), nCounter, and immunohistochemistry. A panel of 46 ancestry-informative markers determined patients' genetic ancestry. RESULTS: The most frequently mutated gene was EGFR (49.6%), followed by TP53 (39.5%), ALK (12.6%), ERBB2 (7.6%), KRAS (5.9%), PIK3CA (1.7%), and less than 1% alterations in RET, NTRK1, MET∆ex14, PDGFRA, and BRAF. Except for TP53 and PIK3CA, all other alterations were mutually exclusive. Genetic ancestry analysis revealed a predominance of European (71.1%), and a higher African ancestry was associated with TP53 mutations. CONCLUSION: Brazilian LCINS exhibited a similar molecular profile to other populations, except the increased ALK and TP53 alterations. Importantly, 73% of these patients have actionable alterations that are suitable for targeted treatments.

KRAS-mutant non-small cell lung cancer (NSCLC) therapy based on tepotinib and omeprazole combination.

BACKGROUND: KRAS-mutant non-small cell lung cancer (NSCLC) shows a relatively low response rate to chemotherapy, immunotherapy and KRAS-G12C selective inhibitors, leading to short median progression-free survival, and overall survival. The MET receptor tyrosine kinase (c-MET), the cognate receptor of hepatocyte growth factor (HGF), was reported to be overexpressed in KRAS-mutant lung cancer cells leading to tumor-growth in anchorage-independent conditions. METHODS: Cell viability assay and synergy analysis were carried out in native, sotorasib and trametinib-resistant KRAS-mutant NSCLC cell lines. Colony formation assays and Western blot analysis were also performed. RNA isolation from tumors of KRAS-mutant NSCLC patients was performed and KRAS and MET mRNA expression was determined by real-time RT-qPCR. In vivo studies were conducted in NSCLC (NCI-H358) cell-derived tumor xenograft model. RESULTS: Our research has shown promising activity of omeprazole, a V-ATPase-driven proton pump inhibitor with potential anti-cancer properties, in combination with the MET inhibitor tepotinib in KRAS-mutant G12C and non-G12C NSCLC cell lines, as well as in G12C inhibitor (AMG510, sotorasib) and MEK inhibitor (trametinib)-resistant cell lines. Moreover, in a xenograft mouse model, combination of omeprazole plus tepotinib caused tumor growth regression. We observed that the combination of these two drugs downregulates phosphorylation of the glycolytic enzyme enolase 1 (ENO1) and the low-density lipoprotein receptor-related protein (LRP) 5/6 in the H358 KRAS G12C cell line, but not in the H358 sotorasib resistant, indicating that the effect of the combination could be independent of ENO1. In addition, we examined the probability of recurrence-free survival and overall survival in 40 early lung adenocarcinoma patients with KRAS G12C mutation stratified by KRAS and MET mRNA levels. Significant differences were observed in recurrence-free survival according to high levels of KRAS mRNA expression. Hazard ratio (HR) of recurrence-free survival was 7.291 (p = 0.014) for high levels of KRAS mRNA expression and 3.742 (p = 0.052) for high MET mRNA expression. CONCLUSIONS: We posit that the combination of the V-ATPase inhibitor omeprazole plus tepotinib warrants further assessment in KRAS-mutant G12C and non G12C cell lines, including those resistant to the covalent KRAS G12C inhibitors.

BID expression determines the apoptotic fate of cancer cells after abrogation of the spindle assembly checkpoint by AURKB or TTK inhibitors.

BACKGROUND: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. METHODS: A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. RESULTS: Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. CONCLUSIONS: The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

Disruptive and Truncating TP53 Mutations Are Associated with African-Ancestry and Worse Prognosis in Brazilian Patients with Lung Adenocarcinoma.

INTRODUCTION: TP53 is the most frequently mutated gene in lung tumors, but its prognostic role in admixed populations, such as Brazilians, remains unclear. In this study, we aimed to evaluate the frequency and clinicopathological impact of TP53 mutations in non-small cell lung cancer (NSCLC) patients in Brazil. METHODS: We analyzed 446 NSCLC patients from Barretos Cancer Hospital. TP53 mutational status was evaluated through targeted next-generation sequencing (NGS) and the variants were biologically classified as disruptive/nondisruptive and as truncating/nontruncating. We also assessed genetic ancestry using 46 ancestry-informative markers. Analysis of lung adenocarcinomas from the cBioportal dataset was performed. We further examined associations of TP53 mutations with patients' clinicopathological features. RESULTS: TP53 mutations were detected in 64.3% (n = 287/446) of NSCLC cases, with a prevalence of 60.4% (n = 221/366) in lung adenocarcinomas. TP53 mutations were associated with brain metastasis at diagnosis, tobacco consumption, and higher African ancestry. Disruptive and truncating mutations were associated with a younger age at diagnosis. Additionally, cBioportal dataset revealed that TP53 mutations were associated with younger age and Black skin color. Patients harboring disruptive/truncating TP53 mutations had worse overall survival than nondisruptive/nontruncating and wild-type patients. CONCLUSION: TP53 mutations are common in Brazilian lung adenocarcinomas, and their biological characterization as disruptive and truncating mutations is associated with African ancestry and shorter overall survival.

Genetic Heterogeneity Underlying Phenotypes with Early-Onset Cerebellar Atrophy.

Cerebellar atrophy (CA) is a frequent neuroimaging finding in paediatric neurology, usually associated with cerebellar ataxia. The list of genes involved in hereditary forms of CA is continuously growing and reveals its genetic complexity. We investigated ten cases with early-onset cerebellar involvement with and without ataxia by exome sequencing or by a targeted panel with 363 genes involved in ataxia or spastic paraplegia. Novel variants were investigated by in silico or experimental approaches. Seven probands carry causative variants in well-known genes associated with CA or cerebellar hypoplasia: SETX, CACNA1G, CACNA1A, CLN6, CPLANE1, and TBCD. The remaining three cases deserve special attention; they harbour variants in MAST1, PI4KA and CLK2 genes. MAST1 is responsible for an ultrarare condition characterised by global developmental delay and cognitive decline; our index case added ataxia to the list of concomitant associated symptoms. PIK4A is mainly related to hypomyelinating leukodystrophy; our proband presented with pure spastic paraplegia and normal intellectual capacity. Finally, in a patient who suffers from mild ataxia with oculomotor apraxia, the de novo novel CLK2 c.1120T>C variant was found. The protein expression of the mutated protein was reduced, which may indicate instability that would affect its kinase activity.

Combinatorial Blood Platelets-Derived circRNA and mRNA Signature for Early-Stage Lung Cancer Detection.

Despite the diversity of liquid biopsy transcriptomic repertoire, numerous studies often exploit only a single RNA type signature for diagnostic biomarker potential. This frequently results in insufficient sensitivity and specificity necessary to reach diagnostic utility. Combinatorial biomarker approaches may offer a more reliable diagnosis. Here, we investigated the synergistic contributions of circRNA and mRNA signatures derived from blood platelets as biomarkers for lung cancer detection. We developed a comprehensive bioinformatics pipeline permitting an analysis of platelet-circRNA and mRNA derived from non-cancer individuals and lung cancer patients. An optimal selected signature is then used to generate the predictive classification model using machine learning algorithm. Using an individual signature of 21 circRNA and 28 mRNA, the predictive models reached an area under the curve (AUC) of 0.88 and 0.81, respectively. Importantly, combinatorial analysis including both types of RNAs resulted in an 8-target signature (6 mRNA and 2 circRNA), enhancing the differentiation of lung cancer from controls (AUC of 0.92). Additionally, we identified five biomarkers potentially specific for early-stage detection of lung cancer. Our proof-of-concept study presents the first multi-analyte-based approach for the analysis of platelets-derived biomarkers, providing a potential combinatorial diagnostic signature for lung cancer detection.

A novel TRMT5 mutation causes a complex inherited neuropathy syndrome: The role of nerve pathology in defining a demyelinating neuropathy.

AIMS: We aim to present data obtained from three patients belonging to three unrelated families with an infantile onset demyelinating neuropathy associated to somatic and neurodevelopmental delay and to describe the underlying genetic changes. METHODS: We performed whole-exome sequencing on genomic DNA from the patients and their parents and reviewed the clinical, muscle and nerve data, the serial neurophysiological studies, brain and muscle MRIs, as well as the respiratory chain complex activity in the muscle of the three index patients. Computer modelling was used to characterise the new missense variant detected. RESULTS: All three patients had a short stature, delayed motor milestone acquisition, intellectual disability and cerebellar abnormalities associated with a severe demyelinating neuropathy, with distinct morphological features. Despite the proliferation of giant mitochondria, the mitochondrial respiratory chain complex activity in skeletal muscle was normal, except in one patient in whom there was a mild decrease in complex I enzyme activity. All three patients carried the same two compound heterozygous variants of the TRMT5 (tRNA Methyltransferase 5) gene, one known pathogenic frameshift mutation [c.312_315del (p.Ile105Serfs*4)] and a second rare missense change [c.665 T > C (p.Ile222Thr)]. TRMT5 is a nuclear-encoded protein involved in the post-transcriptional maturation of mitochondrial tRNA. Computer modelling of the human TRMT5 protein structure suggests that the rare p.Ile222Thr mutation could affect the stability of tRNA binding. CONCLUSIONS: Our study expands the phenotype of mitochondrial disorders caused by TRTM5 mutations and defines a new form of recessive demyelinating peripheral neuropathy.

Mitochondrial developmental encephalopathy with bilateral optic neuropathy related to homozygous variants in IMMT gene.

IMMT gene codes for mitofilin, a mitochondrial inner membrane protein that regulates the morphology of mitochondrial cristae. The phenotype associated with mutations in this gene has not been yet established, but functional studies carried out show that its loss causes a mitochondrial alteration, both in the morphology of the mitochondrial crests and in their function. We present two cousins from an extended highly consanguineous family with developmental encephalopathy, hypotonia, nystagmus due to optic neuropathy. The likely pathogenic homozygous c.895A>G (p.Lys299Glu) variant in the IMMT gene co-segregates with the disease and associates altered mitochondrial cristae observed by electron microscopy.

Extending the clinical phenotype of SPTAN1: From DEE5 to migraine, epilepsy, and subependymal heterotopias without intellectual disability.

Mutations in SPTAN1 gene, encoding the nonerythrocyte αII-spectrin, are responsible for a severe developmental and epileptic encephalopathy (DEE5) and a wide spectrum of neurodevelopmental disorders, as epilepsy with or without intellectual disability (ID) or ID with cerebellar syndrome. A certain genotype-phenotype correlation has been proposed according to the type and location of the mutation. Herein, we report three novel cases with de novo SPTAN1 mutations, one of them associated to a mild phenotype not previously described. They range from (1) severe developmental encephalopathy with ataxia and a mild cerebellar atrophy, without epilepsy; (2) moderate intellectual disability, severe language delay, ataxia and tremor; (3) normal intelligence, chronic migraine, and generalized tonic-clonic seizures. Remarkably, all these patients showed brain MRI abnormalities, being of special interest the subependymal heterotopias detected in the latter patient. Thus we extend the SPTAN1-related phenotypic spectrum, both in its radiological and clinical involvement. Furthermore, after systematic analysis of all the patients so far reported, we noted an excess of male versus female patients (20:9, p = 0.04), more pronounced among the milder phenotypes. Consequently, some protection factor might be suspected among female carriers, which if confirmed should be considered when establishing the pathogenicity of milder genetic variants in this gene.

Volumetric study of brain MRI in a cohort of patients with neurotransmitter disorders.

PURPOSE: Inborn errors of neurotransmitters are rare monogenic diseases. In general, conventional neuroimaging is not useful for diagnosis. Nevertheless, advanced neuroimaging techniques could provide novel diagnosis and prognosis biomarkers. We aim to describe cerebral volumetric findings in a group of Spanish patients with neurotransmitter disorders. METHODS: Fifteen 3D T1-weighted brain images from the International Working Group on Neurotransmitter related Disorders Spanish cohort were assessed (eight with monoamine and seven with amino acid disorders). Volumes of cortical and subcortical brain structures were obtained for each patient and then compared with those of two healthy individuals matched by sex and age. RESULTS: Regardless of the underlying disease, patients showed a smaller total cerebral tissue volume, which was apparently associated with clinical severity. A characteristic volumetric deficit pattern, including the right Heschl gyrus and the bilateral occipital gyrus, was identified. In severe cases, a distinctive pattern comprised the middle and posterior portions of the right cingulate, the left superior motor area and the cerebellum. In succinate semialdehyde dehydrogenase deficiency, volumetric affection seems to worsen over life. CONCLUSION: Despite the heterogeneity and limited size of our cohort, we found novel and relevant data. Total volume deficit appears to be a marker of severity, regardless of the specific neurotransmitter disease and irrespective of the information obtained from conventional neuroimaging. Volumetric assessment of individual brain structures could provide a deeper knowledge about pathophysiology, disease severity and specific clinical traits.

Multiplex Detection of Clinically Relevant Mutations in Liquid Biopsies of Cancer Patients Using a Hybridization-Based Platform.

BACKGROUND: With the advent of precision oncology, liquid biopsies are quickly gaining acceptance in the clinical setting. However, in some cases, the amount of DNA isolated is insufficient for Next-Generation Sequencing (NGS) analysis. The nCounter platform could be an alternative, but it has never been explored for detection of clinically relevant alterations in fluids. METHODS: Circulating-free DNA (cfDNA) was purified from blood, cerebrospinal fluid, and ascites of patients with cancer and analyzed with the nCounter 3 D Single Nucleotide Variant (SNV) Solid Tumor Panel, which allows for detection of 97 driver mutations in 24 genes. RESULTS: Validation experiments revealed that the nCounter SNV panel could detect mutations at allelic fractions of 0.02-2% in samples with ≥5 pg mutant DNA/µL. In a retrospective analysis of 70 cfDNAs from patients with cancer, the panel successfully detected EGFR, KRAS, BRAF, PIK3CA, and NRAS mutations when compared with previous genotyping in the same liquid biopsies and paired tumor tissues [Cohen kappa of 0.96 (CI = 0.92-1.00) and 0.90 (CI = 0.74-1.00), respectively]. In a prospective study including 91 liquid biopsies from patients with different malignancies, 90 yielded valid results with the SNV panel and mutations in EGFR, KRAS, BRAF, PIK3CA, TP53, NFE2L2, CTNNB1, ALK, FBXW7, and PTEN were found. Finally, serial liquid biopsies from a patient with NSCLC revealed that the semiquantitative results of the mutation analysis by the SNV panel correlated with the evolution of the disease. CONCLUSIONS: The nCounter platform requires less DNA than NGS and can be employed for routine mutation testing in liquid biopsies of patients with cancer.

Dihydroartemisinin overcomes the resistance to osimertinib in EGFR-mutant non-small-cell lung cancer.

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), is commonly used to treat EGFR-mutant non-small-cell lung cancer (NSCLC). However, acquired resistance to mutant EGFR (T790M) can evolve following osimertinib treatment. High reactive oxygen species (ROS) levels in lung cancer cells can influence heme levels and have an impact on osimertinib resistance. Here, we found that heme levels were increased in osimertinib resistant EGFR-mutant NSCLC cell lines and plasma heme levels were also elevated in osimertinib-treated EGFR-mutant NSCLC patients. The antimalarial drug dihydroartemisinin (DHA), which has anticancer effects and requires heme, was tested to determine its potential to revert osimertinib resistance. DHA downregulated the expression of heme oxygenase 1 and inhibited cell proliferation in osimertinib-resistant EGFR-mutant NSCLC cells (PC9-GR4-AZD1), which was further enhanced by addition of 5-aminolevulinic acid, protoporphyrin IX and hemin. DHA was synergistic with osimertinib in inhibiting cell proliferation and colony formation of all osimertinib-resistant cell lines tested. Combination treatment with osimertinib and DHA also increased the levels of ROS, downregulated the phosphorylation or protein levels of several RTKs that often are overexpressed in osimertinib-resistant EGFR-mutant NSCLC cells, and inhibited tumor growth without toxicity in a PC9-GR4-AZD1 xenograft mouse model. The results suggest that DHA is able to reverse the resistance to osimertinib in EGFR-mutant NSCLC by elevating ROS level and impair heme metabolism.

Detection of somatic mutations in peritoneal lavages and plasma of endometrial cancer patients: A proof-of-concept study.

Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Although most patients are diagnosed at early stages, 15-20% will relapse despite local treatment. Presently, there are no reliable markers to identify patients with worse outcomes who may benefit from adjuvant treatments, such as chemotherapy, and liquid biopsies may be of use in this setting. Peritoneal lavages are systematically performed during endometrial surgery but little data are available about their potential as liquid biopsies. We analyzed KRAS and PIK3CA mutations in paired surgical biopsies, blood and cytology-negative peritoneal lavages in a cohort of 50 EC patients. Surgical biopsies were submitted to next-generation sequencing (NGS) while circulating-free DNA (cfDNA) purified from plasma and peritoneal lavages was analyzed for KRAS and PIK3CA hotspot mutations using a sensitive quantitative polymerase chain reaction (PCR) assay. NGS of biopsies revealed KRAS, PIK3CA or concomitant KRAS + PIK3CA mutations in 33/50 (66%) EC patients. Of those, 19 cases carried hotspot mutations. Quantitative PCR revealed KRAS and/or PIK3CA mutations in the lavages of 9/19 (47.4%) hotspot EC patients. In contrast, only 2/19 (10.5%) blood samples from hotspot EC patients were positive. Mutations found in cfDNA consistently matched those in paired biopsies. One of the two patients positive in plasma and lavage died in less than 6 months. In conclusion, mutational analysis in peritoneal lavages and blood from early stage EC is feasible. Further studies are warranted to determine if it might help to identify patients with worse prognosis. Human genes discussed: KRAS, KRAS proto-oncogene, GTPase; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

Comutation and exclusion analysis in human tumors: A tool for cancer biology studies and for rational selection of multitargeted therapeutic approaches.

Malignant tumors originate from somatic mutations and other genomic and epigenomic alterations, which lead to loss of control of the cellular circuitry. These alterations present patterns of co-occurrence and mutual exclusivity that can influence prognosis and modify response to drugs, highlighting the need for multitargeted therapies. Studies in this area have generally focused in particular malignancies and considered whole genes instead of specific mutations, ignoring the fact that different alterations in the same gene can have widely different effects. Here, we present a comprehensive analysis of co-dependencies of individual somatic mutations in the whole spectrum of human tumors. Combining multitesting with conditional and expected mutational probabilities, we have discovered rules governing the codependencies of driver and nondriver mutations. We also uncovered pairs and networks of comutations and exclusions, some of them restricted to certain cancer types and others widespread. These pairs and networks are not only of basic but also of clinical interest, and can be of help in the selection of multitargeted antitumor therapies. In this respect, recurrent driver comutations suggest combinations of drugs that might be effective in the clinical setting, while recurrent exclusions indicate combinations unlikely to be useful.

Targeting PKCι-PAK1 signaling pathways in EGFR and KRAS mutant adenocarcinoma and lung squamous cell carcinoma.

INTRODUCTION: p21-activated kinase 1 (PAK1) stimulates growth and metastasis in non-small cell lung cancer (NSCLC). Protein kinase C iota (PKCι) is an enzyme highly expressed in NSCLC, regulating PAK1 signaling. In the present study we explored whether the PKCι-PAK1 signaling pathway approach can be an efficient target in different types of NSCLC cell and mouse models. METHODS: The effect of IPA-3 (PAK1 inhibitor) plus auranofin (PKCι inhibitor) combination was evaluated by cell viability assay, colony formation and western blotting assay, using three types of NSCLC cell lines: EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma with PAK1 amplification. In addition, for clinical availability, screening for new PAK1 inhibitors was carried out and the compound OTSSP167 was evaluated in combination with auranofin in cell and mice models. RESULTS: The combination of IPA-3 or OTSSP167 plus auranofin showed high synergism for inhibiting cell viability and colony formation in three cell lines. Mechanistic characterization revealed that this drug combination abrogated expression and activation of membrane receptors and downstream signaling proteins crucial in lung cancer: EGFR, MET, PAK1, PKCι, ERK1/2, AKT, YAP1 and mTOR. A nude mouse xenograft assay demonstrated that this drug combination strongly suppressed tumor volume compared with single drug treatment. CONCLUSIONS: Combination of IPA-3 or OTSSP167 and auranofin was highly synergistic in EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma cell lines and decreased tumor volume in mice models. It is of interest to further test the targeting of PKCι-PAK1 signaling pathways in EGFR mutant, KRAS mutant and squamous NSCLC patients.

The importance of biochemical and genetic findings in the diagnosis of atypical Norrie disease.

BACKGROUND: Norrie disease (ND) is a rare X-linked disorder characterized by bilateral congenital blindness. ND is caused by a mutation in the Norrie disease pseudoglioma (NDP) gene, which encodes a 133-amino acid protein called norrin. Intragenic deletions including NDP and adjacent genes have been identified in ND patients with a more severe neurologic phenotype. We report the biochemical, molecular, clinical and radiological features of two unrelated affected males with a deletion including NDP and MAO genes. METHODS: Biochemical and genetic analyses were performed to understand the atypical phenotype and radiological findings. Biogenic amines in cerebrospinal fluid (CSF) were measured by high-performance liquid chromatography. The coding exons of NDP gene were amplified by polymerase chain reaction. Multiplex ligation-dependent probe amplification and chromosomal microarray were carried out on both affected males. Computed tomography and magnetic resonance imaging were performed on the two patients. RESULTS: In one patient, the serotonin and catecholamine metabolite levels in CSF were virtually undetectable. In both patients, genetic studies revealed microdeletions in the Xp11.3 region, involving the NDP, MAOA and MAOB genes. Radiological examination demonstrated brain and cerebellar atrophy. CONCLUSIONS: We suggest that alterations caused by MAO deficit may remain during the first years of life. Clinical phenotype, biochemical findings and neuroimaging can guide the genetic study in patients with atypical ND and help us to a better understanding of this disease.

Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients.

BACKGROUND: When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA. METHODS: Our panel ('SiRe') covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice. RESULTS: SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression. CONCLUSIONS: SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.

Identification of ALK, ROS1, and RET Fusions by a Multiplexed mRNA-Based Assay in Formalin-Fixed, Paraffin-Embedded Samples from Advanced Non-Small-Cell Lung Cancer Patients.

BACKGROUND: Anaplastic lymphoma receptor tyrosine kinase (ALK), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1), and ret proto-oncogene (RET) fusions are present in 5%-7% of patients with advanced non-small-cell lung cancer (NSCLC); their accurate identification is critical to guide targeted therapies. FISH and immunohistochemistry (IHC) are considered the gold standards to determine gene fusions, but they have limitations. The nCounter platform is a potentially useful genomic tool for multiplexed detection of gene fusions, but has not been validated in the clinical setting. METHODS: Formalin-fixed, paraffin embedded (FFPE) samples from 108 patients with advanced NSCLC were analyzed with an nCounter-based assay and the results compared with FISH, IHC, and reverse transcription PCR (RT-PCR). Data on response to fusion kinase inhibitors was retrospectively collected in a subset of 29 patients. RESULTS: Of 108 FFPE samples, 98 were successfully analyzed by nCounter (91%), which identified 55 fusion-positive cases (32 ALK, 21 ROS1, and 2 RET). nCounter results were highly concordant with IHC for ALK (98.5%, CI = 91.8-99.7), while 11 discrepancies were found compared with FISH (87.5% concordance, CI = 79.0-92.9). For ROS1, nCounter showed similar agreement with IHC and FISH (87.2% and 85.9%), but a substantial number of samples were positive only by 1 or 2 techniques. Of the 25 patients deriving clinical benefit from fusion kinase inhibitors, 24 were positive by nCounter and 22 by FISH. CONCLUSIONS: nCounter compares favorably with IHC and FISH and can be used for identifying patients with advanced NSCLC positive for ALK/ROS1/RET fusion genes.

Clinical rating scale for pantothenate kinase-associated neurodegeneration: A pilot study.

BACKGROUND: Pantothenate kinase-associated neurodegeneration is a progressive neurological disorder occurring in both childhood and adulthood. The objective of this study was to design and pilot-test a disease-specific clinical rating scale for the assessment of patients with pantothenate kinase-associated neurodegeneration. METHODS: In this international cross-sectional study, patients were examined at the referral centers following a standardized protocol. The motor examination was filmed, allowing 3 independent specialists in movement disorders to analyze 28 patients for interrater reliability assessment. The scale included 34 items (maximal score, 135) encompassing 6 subscales for cognition, behavior, disability, parkinsonism, dystonia, and other neurological signs. RESULTS: Forty-seven genetically confirmed patients (30 ± 17 years; range, 6-77 years) were examined with the scale (mean score, 62 ± 21; range, 20-106). Dystonia with prominent cranial involvement and atypical parkinsonian features were present in all patients. Other common signs were cognitive impairment, psychiatric features, and slow and hypometric saccades. Dystonia, parkinsonism, and other neurological features had a moderate to strong correlation with disability. The scale showed good internal consistency for the total scale (Cronbach's α = 0.87). On interrater analysis, weighted kappa values (0.30-0.93) showed substantial or excellent agreement in 85% of the items. The scale also discriminated a subgroup of homozygous c.1583C>T patients with lower scores, supporting construct validity for the scale. CONCLUSIONS: The proposed scale seems to be a reliable and valid instrument for the assessment of pediatric and adult patients with pantothenate kinase-associated neurodegeneration. Additional validation studies with a larger sample size will be required to confirm the present results and to complete the scale validation testing. © 2017 International Parkinson and Movement Disorder Society.