Treatment Research for Multiple Myeloma (MM)

Treatment Research for Multiple Myeloma (MM) ABSTRACT INTRODUCTION Multiple myeloma (MM) is a hematological neoplasm that appears after transformation and uncontrolled proliferation of plasma cells. MM is characterized by a heterogeneous genetic aberrations and very different clinical outcomes (Avet-Loiseau, . Boyd KD). Although treatments for MM have increased by develop new and more sophisticated therapeutics drugs such an immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs)( Shaji K. Kumar, Raghupathy, Antonio Palumbo) the marked variability of responses indicate that larger studies will be required. Currently, identification of cytogenetic abnormalities is performed by conventional karyotyping and fluorescence in situ hybridization. However, these disorders are not sufficient to explain the malignant phenotype given that are also observed in premalignant states of MM such a monoclonal gammopathy of unknown significance (MGUS) or smoldering myeloma(SM)(Bergsagel, Hideshima). This finding justifies the need for a comprehensive screening of genetic abnormalities in MM patients, which has not been incorporated in the medical workup yet. Recently, the introduction of massive sequencing of patient genome using next-generation sequencing (NGS) technologies has considerably increased the understanding of the biological features of MM. Many works have described the complex and heterogeneous mutational profile of MM patients(bolli nuevo, walker). Whole Exome Sequency (WES) studies in newly diagnosis MM patients have confirmed that more than 50 genes are mutated in the first manifestation of disease (walker). However, only few genes have been detected recurrently mutated at diagnosis, including KRAS, NRAS, BRAF, DIS3, TP53 and FAM46C, and only TP53 mutations have been recurentelly associated with poor survival. In addition, other studies have assessed the clonal evolution over time, pointing out that systemic treatment with chemotherapy may affect the livelihood of some subclones more than others, and thus may influence the tumor evolution over time(Egan JB, bolli, Keats JJ, ) The introduction of targeted studies allows the detection of mutations even with very low allele frequencies at an affordable price, allowing the incorporation of extensive genetic studies to the clinical workup. In the last years, several groups have applied this approach in order to achieve a better patients stratification and prognosis prediction. Although many studies have highlighted the importance of the subclonal landscape in MM and many efforts have been undertaken to stratify patients and predicts their responses, there is no clear relation between sensitives or refractories clones to treatment, and more information about the prognostic impact of this subclonal profile in series of homogeneously treated MM patients is needed. A large number of clinical trial are being carried out with this aim, unify treatments in order to study more effectively the impact of genetic alterations in prognosis. In this work, newly diagnosis MM patient homogenously treated have been genetically characterized using a combination of the most recent techniques, including FISH and ultra-deep targeted sequencing. We applied the highest read depth described in the literature with the aim to detect minority subclones ignored to date. We also integrated these data with the clinical features to find out new patterns of behavior, relate them with survival and reveal new insight into the complexity of clonal and subclonal architecture of MM. Patient samples Samples were taken from the available 79 newly diagnosed MM patient enrolled in the clinical trial GEM10MAS65 (registered at www.clinicaltrials.gov as #NCT01237249). This is a phase III trial where patients older than 65 years were randomly assigned between two treatments arms: sequential melphalan/prednisone/Velcade (MPV) followed by Revlimid/low dose dexamethasone (Rd) versus alternating melphalan/prednisone/Velcade (MPV) with Revlimid/low dose dexamethasone (Rd). Progression free survival (PFS) and overall survival (OS) were measured from the starting date of the treatment. The median time to progression was 26.4 months with a median follow up of 31.5 months. Targeted sequencing and mutation calling Positive plasma cells CD138 were isolated from bone marrow aspirates and DNA was extracted using AllPrep DNA/RNA mini kit (Quiagen). Only 20 ng of DNA were used to prepare libraries using Ampliseq Library Kit 2.0. We also sequenced DNA from the 15 available CD138 negative fractions in order to filter out potential artifacts and corroborate detection sentivity. Samples were sequenced using Ion Torrent platform (IonProton, Thermofisher, Carlsbad, CA, USA) using the M3P gene panel (Mayo Clinic, Arizona). This panel spreads out over 77 genes frequently mutated in MM, which are related to critical pathophysiological pathways, associated to drug resistance or targetable with molecular drugs [paper mayo kortum etal]. Quality filter and alignments was performed using Torrent Suit software (Life Technologies) Single nucleotide variants were calling and annotated using Ion Reporter software applying in-house modifications in call variants process. Variants listed in Single Nucleotide Polimorph ism database (dbSNP, http://www.ncbi.nlm.nih.gov/SNP/) were excluded from samples without germline available, as well as variants that were detected in multiple samples. In addition, to test the ability of the workflow previously described, we applied a novel bioinformatics pipeline developed by Spanish National Cancer Research Centre (CNIO). All reported mutations were detected by both bioinformatics approaches. The integrative genomic viewer (IGV) was used to visualize the read alignments, single variants and correct sequencing errors due to homopolymer regions. Statistical analysis All statistical analysis was performed using the statistical environment R. Correlation coefficients between mutated genes and cytogenetic aberrations was assessed and plotted using corrplot (https://cran.r-project.org/web/packages/corrplot/). Differences in survival were tested using the log-rank test. Cox proportional hazard regression was employed to obtain hazards ratios (HR) and evaluated at 5% of significance level. A second approach called LASSO (least absolute shrinkage and selection operator) was performed to detect relevant variables among clinical, cytogenetic and mutated genes.   Further details can be found in Data Supplement. RESULTS Mutated genes and altered pathways (cambiar tà ­tulo) We sequenced 79 tumor samples with a mean coverage depth of 1600X. The minimum coverage of the detected variants was 60X and the average coverage 370X. We identified 170 nonsynonymous missense/nonsense/stoploss single variants, 81 of them (48%) were predicted pathogenic by Sift and Polyphen and 61 (36%)   have been described in COSMIC data base.   85% of patients harbored at least 1 mutation with a median of 2.1 mutations per patient. We detected mutations in 53 genes (Figure1), although 6 genes accounted the 39% of the total number of mutations; KRAS= 21.5%, DIS3= 19%, NRAS= 16.5%, BRAF= 10.1%, TP53= 8.8% and ATM= 7.6% of the patients.  Ã‚   48% of patients (38/79) presented at least one mutation in genes envolved in RAS/MAPK pathway, being the most frequently mutated pathway. 72 and 100% of variants in KRAS and NRAS respectively were detected in the hotspot codons 12, 13 and 61, and the targetable V600E BRAF mutation was detected in 1 patient. (Figure 2 supplemental?). NFKB p athway was the second most frequently mutated in our cohort, accounting for the 15% of all mutations distributed in 25 % of the patients (19/79). This pathway included TRAF3 (5 mutations, one nonsense and 4 missense) and TRAF2 (3 missense mutation) all of them predicted pathogenic by Sift and Polyphen. Other pathways importantly altered in the cohort were MYC in 11% of patients (9/79), cereblon and ciclyn both in 9% of patients (7/79). Multiple mutations within the same gene were observed in 11 patients: 4 of whom harbored 2 and 3 mutations within DIS3 (patient 1-24= Glu501Lys and Phe120Leu at 8 and 53 % of VRF, patient 2-236= Asp487His and Asp479Glu at 4 and 21 of VRF respectively, patient 3 321= Tyr753Asn and Glu126Lys at 2 and 58 % of VRF respectively and patient 4-42 = Arg820Trp, Gly249Glu and at 14, 20 and 24% of VRF respectively). Other 2 patients showed 2 and 3 mutation in KRAS (patient 5-168= Gly13Asp and Gln61His at 9 and 13 %of VRF and patient 6-269 = Tyr71Asp, UTR3 in exon 6 and Gln61Glu at 3, 15 and 37 % of VRF respectively; 2 patients with 2 mutation in NRAS(patient 7-177= .Gln61Lys   and Gly12Ala at 5 and 12% of VRF respectively and patient 8-257=   Gln61Glu   and Gly12Ala at 5 and 6% of VRF respectively), one patient with 3 mutations in MAX (patient 9-190= Arg36Lys , Arg35Leu and Glu32Val at 10, 20 and 26 %of VRF), one patient with 3 mutations in TRAF3 (patient 10-40 = Lys453Asn, His136Tyr   and Phe445Leu 8, 11 and1 3% of VRF) and one patient with 2 mutation in TP53 (patient 11-40 = Asp208Val   and Glu204Ter at 35 and 36% of VRF respectively). Variant Read Frequency study The VRF found in our cohort were diverse.   We detected 50% of variants (85/170) below 25% of VRF and 27% (46/170) below 10% (Figure 2). KRAS (n=5) DIS3 (n=5), BRAF (n=4), NRAS (n=4), and TP53 (n=3) were the genes that harbored the largest number of low frequency mutations (Figure 2). KRAS, NRAS, BRAF and TP53 mutations were, in all cases, lower than 50% of VRF while DIS3 showed mutations in a broad range (from 2 to 85%)(Fig3). ). Most of DIS3 mutations with VRF

Cyber Forensics: Development of a Case Hypothesis Essay

Throughout time past, forensic science disciplines have helped solved numerous crime investigations and it has given impelling testimony in the area of court trials. In order to reduce the sagacity of siding or bias situation and avoidance in prosecuting innocent victims, it is important to analyse, validate and have proper presentation of digital evidence in the context of cyber forensics examinations. [1] (Noblett et al, 2000) in this essay we will discuss on various topics that describe, explain or illustrate on issues such as the processes that assist in the development of a case hypothesis and as well as alternative hypothesis. It will also cover the processes in which how validation check and test are conducted to determine the accuracy of the digital evidence. Furthermore, we will look into the deductive, inductive and abductive reasoning in the field of cyber forensics. Lastly, the essay will also cover on the processes that would improve the communication and presentation of case analysis to the solicitors and courts. INTRODUCTION Before the term cyber forensics was introduced in the late 1960s, most crimes are formally solved using traditional forensic science disciplines. Before the first PC computer was invented, crimes in those days were not as complicated as compared to today. In this introduction section, we will distinguish between what is forensic science and how it is different from cyber forensics. Forensic science depends on the capability of the research scientists to develop a case report based on the outcome of a scientific review. For instance , a DNA report analysis of a murder case can be undertaken without the prior knowledge of the victim’s name or exact situation of the crime.[2] (Chakraborty, R.1990) On the contrary, cyber forensics science’s main focus is driven on information discovered during the investigation. However the challenge lies in the search hunt of valid and admissible evidence in the media storage of a computer. The average storage capacity of a PC is approxima tely 300-500 Gigabytes; therefore it is tough to totally scan through every single file stored on a suspect’s computer system, let alone those computer networks. [3] [Casey, E. (2004] PROCESSES THAT ASSIST TO DEVELOP CASE HYPOTHESIS /ALTERNATE HYPOTHESIS Before  we develop a case hypothesis or alternate hypothesis, there are several procedures and guidelines that a forensic investigator must follow and do. Firstly, the investigators must construct a hypothesis of the occurrence which is based on the study of the evidence. On the other hand, the degree of rigidity of this hypothesis also relies upon the type of investigation. For instance, an Interpol police investigation would require the preparation of a detailed hypothesis with discreet and meticulous proper documentation to support specimens identified during the examination.[4] [Ó Ciardhuà ¡in, 2004]In the case of a police investigation , the hypothesis will be presented before a jury however the hypothesis drawn in a company will be handled by the management. Technically the hypothesis will be verified and an alternate hypothesis as well as supporting evidence will be presented before a jury. The investigators will need to affirm the legitimacy of their hypothesis and protect it against any critics or provocation. In the event if the challenge is successful, there will be a need to backtrack to the earlier stages to collect and search for more evidences so as to construct a better hypothesis. Talking to the experts AKA† Hot tubbing† is widely used for coexisting evidence. This process involves the court to put several expert witnesses on the stand together which will in turn saves much time and resources. Moreover, there are two main types of witnesses’ testimony at a trial, deposition or hearing. They are technical or scientific witness testimony and expert witness testimony. [5][Enfinger, 2006] As for technical or scientific witness, the investigator would need to present details of evidence that were discovered during the investigation. They would be asked to describe what was discovered and how it was acquired. During the compilation of the evidence , the investigator must ensure that the evidence collected must be legal and done appropriately with the permission of the owner and the suspect as well as a search warrant or hot pursuit. Also, it is essential that exculpatory and inculpatory evidence is presented. [6] [Cohen, 2006] On the other hand , the investigator would draft out the chains of events that have certain connections and linkage to form the chain of custody which is basically a documentation or paper trail displaying the seizure, control, transfer, analysis, custody and deposition of physical or digital. Apart from the chain of custody, there is another process known as the chain of Inference which is also referred as concatenate inferences.  These inferences between the weak and the strong ones build upon one another until they reduce the gap between the defendant and the conclusion to a manageable distance. The concatenate inferences process may be interpreted by fabricating a hypothetical scenario. The purpose of constructing a chain of inferences is to convince a fact finder that the desired conclusion is the most plausible sequence of events. On top of this, it is also vital to internalise the difference between evidence and inference before the development of a hypothesis or the reconstruction of the crime scene. With that comes the formation of crime scene timelines which is an efficient method to derive a conclusion. It is a graphical chart that illustrates the activity time line of crime scene sorted based on the sequence of events. These log entries displayed a unique chain of events that culminate in the incident which is a closer step towards proving a case. [8] [Stephenson, 2000] Another important process is testing, analysing and reporting. Testing is to ensure that all evidence both physical and electronic gathered must be verified and gone through quality check by scientific personnel to affirm the originality [without contamination] as well as how this proof of evidence would be of any aid to solve the crime. Analysis deals with what are the issues identify and intention of the crime act and for each issue how it can be addressed, documented, tested and verified. Lastly this analysis will be written down and documented as a report. [9] [Robert F. Winch and Donald T. Campbell, 1969]

What Everyone Is Saying About Sample of a Research Paper Is Dead Wrong and Why

What Everyone Is Saying About Sample of a Research Paper Is Dead Wrong and Why It is preferable to use total sentence outline structure, if your paper ought to be formatted in MLA. The papers might be rejected if they don't apply the APA format. A superb outline is a significant element in writing a very good paper. Completing an MLA outline will make sure your research paper format is accurate. Research Paper isn't a task for a single day. Regardless of what research paper outline you're considering, you will want to consider how your research is going to be backed up. Research is a good example of the way in which a gradual focusing of research will help to define how to compose a hypothesis. The Bizarre Secret of Sample of a Research Paper The best type of essay starts with the ideal form of topic, so take time to choose something which works for you. 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The Medias Dependence On Technology - 1510 Words

The Screens of Evil Since the beginning of time, humans have strived to live simpler lives. We have spent generations, creating innovations within technology to ensure our lives would always be more simplistic than those who lived centuries before us. People now have the ability to update their Facebook status’, map the human genome to target strands of DNA that can be disastrous to one’s health, send out amber alerts nationwide, and create plants that are resistant to pesticides. Innovation in technology has led to advancements in many fields such as medicine, law enforcement, and entertainment which has inevitably changed the way society operates. However, with the abundance of technology available, society has developed an unhealthy†¦show more content†¦In Carolyn Gregoire’s article â€Å" Babies And iPads Might Not Be Such A Great Combination After All†, she discusses a recommendation from the American Academy of Pediatrics that suggests children under the age of two should have little exposure to games and media. Children’s brains grow rapidly during the first years of life and â€Å"...young children learn best by interacting with people, not screens† (Gregoire). Unfortunately, it has become common for parents to rely on technology to distract or â€Å"teach† their children. However, Loren Frank, a psychologist at the University of California, explains that when the brain is being constantly stimulated you begin to prevent the learning process. The psychologist also mentions that allowing our brains to have downtime is essential for solidifying our experiences and developing them into permanent memories (Greenblatt). Studies have shown that when children are able to play with materials that help build foundations for understanding concepts and skills, they are given the ability to make solid connections that create pathways that stimulate the brain (Carlsson-Paige). Moreover, it is not just the youth that is being targeted by technology, but the human population at large. A vital aspect of our daily lives is communication. The internet, and the social