Optimized pipeline of MuTect and GATK tools to improve the detection of somatic single nucleotide polymorphisms in whole- exome sequencing data

Background: Detecting somatic mutations in whole exome sequencing data of cancer samples has become a popular approach for profiling cancer development, progression and chemotherapy resistance. Several studies have proposed software packages, filters and parametrizations. However, many research groups reported low concordance among different methods. We aimed to develop a pipeline which detects a wide range of single nucleotide mutations with high validation rates. We combined two standard tools – Genome Analysis Toolkit (GATK) and MuTect – to create the GATK-LODN method. As proof of principle, we applied our pipeline to exome sequencing data of hematological (Acute Myeloid and Acute Lymphoblastic Leukemias) and solid (Gastrointestinal Stromal Tumor and Lung Adenocarcinoma) tumors. We performed experiments on simulated data to test the sensitivity and specificity of our pipeline. Results: The software MuTect presented the highest validation rate (90 %) for mutation detection, but limited number of somatic mutations detected. The GATK detected a high number of mutations but with low specificity. The GATK-LODN increased the performance of the GATK variant detection (from 5 of 14 to 3 of 4 confirmed variants), while preserving mutations not detected by MuTect. However, GATK-LODN filtered more variants in the hematological samples than in the solid tumors. Experiments in simulated data demonstrated that GATK-LODN increased both specificity and sensitivity of GATK results. Conclusion: We presented a pipeline that detects a wide range of somatic single nucleotide variants, with good validation rates, from exome sequencing data of cancer samples. We also showed the advantage of combining standard algorithms to create the GATK-LODN method, that increased specificity and sensitivity of GATK results. This pipeline can be helpful in discovery studies aimed to profile the somatic mutational landscape of cancer genomes

Five years of ocrelizumab in relapsing multiple sclerosis : OPERA studies open-label extension

Objective: To assess over 3 years of follow-up the effects of maintaining or switching to ocrelizumab (OCR) therapy on clinical and MRI outcomes and safety measures in the open-label extension (OLE) phase of the pooled OPERA: I/II studies in relapsing multiple sclerosis. Methods:After 2 years of double-blind, controlled treatment, patients continued OCR (600 mg infusions every 24 weeks) or switched from interferon (IFN)-β-1a (44 g 3 times weekly) to OCR when entering the OLE phase (3 years). Adjusted annualized relapse rate, time to onset of 24-week confirmed disability progression (CDP)/improvement (CDP), brain MRI activity (gadolinium-enhanced and new/enlarging T2 lesions), and percentage brain volume change were analyzed. Results:Of patients entering the OLE phase, 88.6% completed year 5. The cumulative proportion with 24-week CDP was lower in patients who initiated OCR earlier vs patients initially receiving IFN-β-1a (16.1% vs 21.3% at year 5; p = 0.014). Patients continuing OCR maintained and those switching from IFN-β-1a to OCR attained near complete and sustained suppression of new brain MRI lesion activity from years 3-5. Over the OLE phase, patients continuing OCR exhibited less whole brain volume loss from double-blind study baseline vs those switching from IFN-β-1a (-1.87% vs-2.15% at year 5; p < 0.01). Adverse events were consistent with past reports and no new safety signals emerged with prolonged treatment.ConclusionCompared with patients switching from IFN-β-1a, earlier and continuous OCR treatment up to 5 years provided sustained benefit on clinical and MRI measures of disease progression.Classification of evidenceThis study provides Class III evidence that earlier and continuous treatment with OCR provided sustained benefit on clinical and MRI outcomes of disease activity and progression compared with patients switching from IFN-β-1a. The study is rated Class III because of the initial treatment randomization disclosure that occurred after inclusion in OLE.Clinical trial identifiersNCT01247324/NCT01412333

Evaluation of cerebral hemodynamic in patients with subarachnoid hemorrhage: the role of perfusion computed tomography

Scopo dello studio - L'emorragia subaracnoidea da rottura di un aneurisma intracranico \ue8 una patologia devastante con un tasso di mortalit\ue0 prossimo al 50%, cui si aggiunge un ulteriore 30% di disabilit\ue0. L'ischemia cerebrale in fase subacuta e gli infarti che ne possono conseguire sono la principale causa di deficit a lungo termine. Le alterazioni dell'emodinamica cerebrale dovute all'emorragia subaracnoidea sono il risultato dell'interazione di vari fattori tra cui l'impatto iniziale sull'encefalo, l'alterazione dell'autoregolazione e il vasospasmo cerebrale. In questo studio abbiamo valutato le alterazioni della perfusione cerebrale e il loro andamento nel tempo nelle prime due settimane dopo l'emorragia, utilizzando la Perfusion Computed Tomography Scan (PCT scan). Inoltre abbiamo cercato di identificare i parametri di perfusione associati con lo sviluppo di vasospasmo cerebrale e infarto. Metodo - I quaranta pazienti studiati sono stati sottoposti a PCT scans secondo prestabiliti intervalli di tempo dall'emorragia (0-3 giorni, 4-7 giorni, 8-11 giorni e tardivo). Sono state effettuate un numero complessivo di 92 Perfusion CT scans (2.3 per paziente) e sono stati analizzati i territori delle arterie ACA e MCA. Secondo l'andamento clinico i pazienti sono stati divisi 3 gruppi: 22 pazienti che non hanno sviluppato vasospasmo e 18 pazienti che hanno sviluppato vasospasmo, con infarto cerebrale in 8 di essi. La perfusione globale \ue8 stata studiata utilizzando la media (\uf0b1 DS) ottenuta dai valori di ciascun parametro (CBV, CBF and MTT) in ciascun gruppo e in ciascun intervallo di tempo. Abbiamo quindi analizzato i cambiamenti nel tempo di CBV, CBF and MTT in ciascun gruppo e abbiamo confrontato i valori di CBV, CBF e MTT di ciascun gruppo in ogni intervallo di tempo. Risultati- Nel corso delle prime due settimane dall'emorragia i pazienti senza vasospasmo hanno modesti cambiamenti dei parametri di perfusione e i valori nei pazienti con vasospasmo sintomatico sono stabili. Al contrario, nei pazienti con vasospasmo sintomatico e infarto cerebrale, CBV and CBF raggiungono il valore massimo nell'intervallo tra 4 e 7 giorni per poi diminuire nei giorni successivi mentre il valore di MTT si mantiene stabile nei primi due intervalli. Considerando che tutti i pazienti sono stati sottoposti ad una moderata ipervolemia subito dopo l'esclusione dell'aneurisma e che le alterazioni della perfusione indicate sono presenti solo nei pazienti con vasospasmo sintomatico e infarto cerebrale, esse possono essere interpretate come l'espressione dell'intervento emodinamico in pazienti che hanno un'alterazione dell'autoregolazione prima dell'insorgenza di vasospasmo. Questi pazienti hanno una riduzione del valore di CBV e una riduzione significativa del valore di CBF (p=0.016) nell'intervallo tra 8 e 11 giorni e nell'intervallo tardivo il valore di CBF si mantiene significativamente inferiore rispetto a quello nell'intervallo tra 4 e 7 giorni (p=0.044). Nell'intervallo tra 8 e 11 giorni, se confrontati con i pazienti senza vasospasmo, essi hanno un valore di CBF significativamente inferiore (p=0.035) inoltre il loro valore di MTT raggiunge il massimo tra 8 e 11 giorni ed \ue8 significativamente superiore (p=0.031) rispetto ai pazienti senza vasospasmo. Conclusioni - I nostri risultati supportano l'ipotesi che i pazienti con vasospasmo sintomatico e infarto cerebrale hanno un'alterazione dell'autoregolazione cerebrale presente gi\ue0 prima della comparsa del vasospasmo e che essi possono essere studiati con la Perfusion CT scan.Background and Purpose - Subarachnoid hemorrhage due to the rupture of an intracranial aneurysm (aSAH) is a devastating disease with a mortality rate of nearly 50% and with an additional 30% of patients requiring long-term care. Delayed cerebral ischemia and subsequent cerebral infarction are the leading causes of poor outcome. The alterations of cerebral hemodynamic due to aSAH are caused by the interaction of many mechanisms including the early impact of bleeding on the brain, the impairment of cerebral autoregulation and the cerebral vasospasm. In this study we used the Perfusion Computed Tomography Scan (PCT scan) to evaluate the time course of the cerebral perfusion changes over the first two weeks after bleeding. We also attempted to identify the parameters of the perfusion that were associated with the development of cerebral vasospasm and infarction. Methods - Forty patients with aSAH underwent PCT scans according to regular intervals of time from SAH (0-3 days, 4-7 days, 8-11 days and later). A total number of 92 Perfusion CT studies (2.3 per patient) were carried out and ACA and MCA territory were studied. Out of 40 patients, 22 patients did not develop symptomatic vasospasm and 18 patients developed symptomatic vasospasm, with cerebral infarction in 8 of them. We analyzed the global cerebral perfusion using the mean value (\uf0b1 SD) obtained by the average of the values of each parameter (CBV, CBF and MTT) in each group of patients and in the established intervals of time. The changes of CBV, CBF and MTT over time for each group of patients were analysed and the values of the three groups in each interval of time were compared. Results - Patients without vasospasm have little changes, and patients with symptomatic vasospasm have no changes of the perfusion values throughout the study. On the contrary, in patients with symptomatic vasospasm and cerebral infarction, CBV and CBF reach the maximum value in the interval between 4 and 7 days and decrease in the following days and MTT is stable in the first two intervals of time. These values can represent the effect of the hemodynamic intervention in patients who have an impairment of cerebral autoregulation in a stage before the onset of vasospasm. The reason of this is that a preventive mild volume expansion was started immediately after the aneurysm treatment in all patients. These patients have a decrease of CBV value and a significant decrease of CBF value (p=0.016) between 8 and 11 days and in the later interval the CBF value is persistently significantly lower than in the interval between 4 and 7 days (p=0.044). Between 8 and 11 days, in the comparison with the group of patients without vasospasm, they have a significantly lower value of CBF (p=0.035). Moreover the MTT reaches the maximum value between 8 and 11 days and it is significantly higher (p=0.031) than in patients without vasospasm in the same interval of time. Conclusions - Our results suggest that patients who experienced ischemic deficits and cerebral infarction have an impairment of cerebral autoregulation before the onset of vasospasm and that they can be studied using the Perfusion CT scan

Risk of requiring a walking aid after 6.5 years of ocrelizumab treatment in patients with relapsing multiple sclerosis: Data from the OPERA I and OPERA II trials

BACKGROUND AND PURPOSE: Requiring a walking aid is a fundamental milestone in multiple sclerosis (MS), represented by an Expanded Disability Status Scale (EDSS) score ≥6.0. In the present study, we assess the effect of ocrelizumab (OCR) on time to EDSS score ≥6.0 in relapsing MS. METHODS: Time to EDSS score ≥6.0 confirmed for ≥24 and ≥48 weeks was assessed over the course of 6.5 years (336 weeks) in the double-blind period (DBP) and open-label extension (OLE) period of the OPERA I (NCT01247324) and OPERA II (NCT01412333) studies. RESULTS: Time to reach EDSS score ≥6.0 was significantly delayed in those initially randomized to OCR versus interferon. Over 6.5 years, the risk of requiring a walking aid confirmed for ≥24 weeks was 34% lower among those who initiated OCR earlier versus delayed treatment (average hazard ratio [HR] DBP + OLE 0.66, 95% confidence interval [CI] 0.45-0.95; p = 0.024); the risk of requiring a walking aid confirmed for ≥48 weeks was 46% lower (average HR DBP+OLE 0.54, 95% CI 0.35-0.83; p = 0.004). CONCLUSION: The reduced risk of requiring a walking aid in earlier initiators of OCR demonstrates the long-term implications of earlier highly effective treatment

Risk of requiring a walking aid after 6.5&nbsp;years of ocrelizumab treatment in patients with relapsing multiple sclerosis: Data from the OPERA I and OPERA II trials.

Background and purposeRequiring a walking aid is a fundamental milestone in multiple sclerosis (MS), represented by an Expanded Disability Status Scale (EDSS) score ≥6.0. In the present study, we assess the effect of ocrelizumab (OCR) on time to EDSS score ≥6.0 in relapsing MS.MethodsTime to EDSS score ≥6.0 confirmed for ≥24 and ≥48&nbsp;weeks was assessed over the course of 6.5&nbsp;years (336&nbsp;weeks) in the double-blind period (DBP) and open-label extension (OLE) period of the OPERA I (NCT01247324) and OPERA II (NCT01412333) studies.ResultsTime to reach EDSS score ≥6.0 was significantly delayed in those initially randomized to OCR versus interferon. Over 6.5&nbsp;years, the risk of requiring a walking aid confirmed for ≥24&nbsp;weeks was 34% lower among those who initiated OCR earlier versus delayed treatment (average hazard ratio [HR] DBP&nbsp;+&nbsp;OLE 0.66, 95% confidence interval [CI] 0.45-0.95; p&nbsp;=&nbsp;0.024); the risk of requiring a walking aid confirmed for ≥48&nbsp;weeks was 46% lower (average HR DBP+OLE 0.54, 95% CI 0.35-0.83; p&nbsp;=&nbsp;0.004).ConclusionThe reduced risk of requiring a walking aid in earlier initiators of OCR demonstrates the long-term implications of earlier highly effective treatment

Association of Higher Ocrelizumab Exposure With Reduced Disability Progression in Multiple Sclerosis

Background and objectivesOcrelizumab improved clinical and MRI measures of disease activity and progression in three phase 3 multiple sclerosis (MS) studies. Post hoc analyses demonstrated a correlation between the ocrelizumab serum concentration and the degree of blood B-cell depletion, and body weight was identified as the most influential covariate on ocrelizumab pharmacokinetics. The magnitude of ocrelizumab treatment benefit on disability progression was greater in lighter vs heavier patients. These observations suggest that higher ocrelizumab serum levels provide more complete B-cell depletion and a greater delay in disability progression. The current post hoc analyses assessed population exposure-efficacy/safety relationships of ocrelizumab in patients with relapsing and primary progressive MS.MethodsPatients in OPERA I/II and ORATORIO were grouped in exposure quartiles based on their observed individual serum ocrelizumab level over the treatment period. Exposure-response relationships were analyzed for clinical efficacy (24-week confirmed disability progression (CDP), annualized relapse rate [ARR], and MRI outcomes) and adverse events.ResultsOcrelizumab reduced new MRI lesion counts to nearly undetectable levels in patients with relapsing or primary progressive MS across all exposure subgroups, and reduced ARR in patients with relapsing MS to very low levels (0.13-0.18). A consistent trend of higher ocrelizumab exposure leading to lower rates of CDP was seen (0%-25% [lowest] to 75%-100% [highest] quartile hazard ratios and 95% confidence intervals; relapsing MS: 0.70 [0.41-1.19], 0.85 [0.52-1.39], 0.47 [0.25-0.87], and 0.34 [0.17-0.70] vs interferon β-1a; primary progressive MS: 0.88 [0.59-1.30], 0.86 [0.60-1.25], 0.77 [0.52-1.14], and 0.55 [0.36-0.83] vs placebo). Infusion-related reactions, serious adverse events, and serious infections were similar across exposure subgroups.DiscussionThe almost complete reduction of ARR and MRI activity already evident in the lowest quartile, and across all ocrelizumab-exposure groups, suggests a ceiling effect. A consistent trend of higher ocrelizumab exposure leading to greater reduction in risk of CDP was observed, particularly in the relapsing MS trials, and was not associated with a higher rate of adverse events. Higher ocrelizumab exposure may provide improved control of disability progression by reducing disease activity below that detectable by ARR and MRI, and/or by attenuating other B-cell-related pathologies responsible for tissue damage.Classification of evidenceThis analysis provides Class III evidence that higher ocrelizumab serum levels are related to greater reduction in risk of disability progression in patients with multiple sclerosis. The study is rated Class III because of the initial treatment randomization disclosure that occurred after inclusion in the open-label extension.Trial registration informationClinicalTrials.gov Identifier: NCT01247324 (OPERA I), NCT01412333 (OPERA II), and NCT01194570 (ORATORIO)

Co-occurrence of alterations in the DNA damage repair genes synergize with uncontrolled proliferation and associate with very-poor prognosis in acute myeloid leukemia patients

Abstract Partner and localizer of BRCA2 (PALB2) plays a key role in the DNA damage repair (DDR) by recruiting BRCA1, BRCA2 and RAD51. Alterations in PALB2 were described in hereditary breast cancer and Fanconi Anemia (FA). Little is known in Acute Myeloid Leukemia (AML). Aim of the study is to define the frequency and interplay of PALB2 alterations with patterns of somatic mutations and copy number variants (CNVs) in AML. We genotyped 233 AML samples by Single Nucleotide Polymorphism array (SNP 6.0 and Cytoscan HD, Affymetrix) to detect CNVs. We also performed Whole Exome Sequencing (WES, Illumina) of 56 cases to detect single nucleotide variants and small indels (MuTect and Varscan 2.0). Differences in survival were assessed by Kaplan-Meier survival analysis and Breslow test. We detected PALB2 loss in 12/233 patients (5%), with a minimal common region of deletion of 6.6 Kb including exon 12, which encodes for the domain of interaction with RAD51, BRCA2 and POLH. PALB2 deletion were correlated with CN loss of MYH11, CREBBP, PLK1 and FANCA on chromosome (chr) 16 (p≤.005), deletions of TP53, NF1 and BRCA1 on chr 17 (p≤.004), 5q deletions (p≤.001) and loss of XPO1 on chr 2 (p≤.006). PALB2-loss patients were enriched for alterations in genes involved in the protein kinase pathway (p=5×10-4), JUN kinase activity (p=.0006) and, notably, in genes belonging to the chromosome breakage pathway (p=.001; TP53, BRCA1 and BRCA2). To identify mutations that co-operates with CN alterations, we integrated WES data of 56 AML patients. No mutations in PALB2 were detected. However, we identified mutations in other DDR genes, including FANCE, BRCA2, TP53 and BRCA1. Of note, alterations in the DDR genes frequently co-occurred with both mutations and CN loss of TP53, leading to homozygous loss of function of TP53. In addition, KRAS, IDH1, TET2 and BCOR mutations were mutually exclusive with PALB2 loss. Preliminary gene expression profiling data revealed a 2-fold upregulation of FANCA in samples with DDR genes alterations compared with patients with no alterations (p=.04), while the other genes of the pathway were not affected. Finally, patients harboring PALB2 loss had a poorer prognosis compared complex karyotype cases (p=.021). We here define a new subset of AML patients, characterized by the synergistic loss of key DDR genes, which may in turn result in FANCA over-expression as a mechanism to overcome the DDR deficit. Our data suggest that the leukemogenic process associated PALB2 loss integrates defects in the DDR together with enforced proliferative stimuli leading to uncontrolled proliferation of cells, which potentially accumulate genetic lesions. These results may help improve patient stratification and define ad hoc therapeutic strategies for this aggressive leukemia type. Supported by: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Antonella Padella, Giorgia Simonetti, Maria Chiara Fontana, Marco Manfrini, Giovanni Marconi, Anna Ferrari, Italo Faria do Valle, Marianna Garonzi, Cristina Papayannidis, Eugenia Franchini, Elisa Zuffa, Viviana Guadagnuolo, Samantha Bruno, Andrea Ghelli Luserna di Rorà, Emanuela Ottaviani, Daniel Remondini, Massimo Delledonne, Giovanni Martinelli. Co-occurrence of alterations in the DNA damage repair genes synergize with uncontrolled proliferation and associate with very-poor prognosis in acute myeloid leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4671. doi:10.1158/1538-7445.AM2017-4671</jats:p

Additional file 1: of Optimized pipeline of MuTect and GATK tools to improve the detection of somatic single nucleotide polymorphisms in whole-exome sequencing data

Supplementary information. A.xls file including supplementary tables. (XLS 25 kb