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A dad who started losing his hair aged 17 bought his son a £3,000 hair transplant for his 18th birthday so he wouldn't go through the same pain.
When Owen Bigwood started losing his hair at 17, his dad Roger, 43, totally sympathised, after having gone through the same experience at an identical age.
But Owen, 19, said the reality TV show TOWIE – featuring highly-groomed young people from the county – had thrown Essex men into the spotlight, making him horribly self-conscious about his thinning crown.
And after booking in Owen for the operation, Roger decided to have one as well so the pair could go through it together.
Owen explained: "Growing up in Little Baddow, near Chelmsford, Essex, where everyone's trying to look their best, made me really aware of going bald.
"I'd think, 'I can't have my hair cut like that, like my mates,' because I had a high hairline, which looked like the McDonald's M!"
"My mates would say they didn't notice my hair, but I hated it. It affected my confidence.
"Chatting to people – especially to girls - I'd feel like the old-looking bald guy.
"Since my hair transplant in March, I already have more confidence."
Owen, who studied business and entrepreneurship at college, and Roger both have male pattern baldness – the most common form of hair loss, which is believed to be genetic.
It was highlighted by footballer Wayne Rooney, who had a hair transplant in 2011.
But it was Owen's mum, Claire, 44, who assists her husband with his businesses, who gave him the idea of having a transplant, after seeing how battered his confidence had been by his baldness.
She was inspired by reading about how others had had the procedure.
He said: "I would brush my hair to cover the bald spots so it didn't look so bad.
"And on windy days I'd wear hats to keep my hair down.
"My mum bought me hair-thickening shampoo and I used it for a few months, but I didn't see a difference.
"When Mum told me on my 18th birthday, on June 15, 2016, I could have a transplant, I was thrilled. It felt like all I'd ever wanted.
"Since having it done, my bald McDonalds arches are filling in with new hair."
Meanwhile, Roger, who has four sons and two daughters - Owen, Seth, 15, Asher, nine, and Theo, six, and two daughters, Gabriella, 18, and Roseanna, 12 – says it made sense for him to have a transplant, too.
He explained: "I knew how Owen was feeling, as I'll never forget my sister cutting my hair when I was the same age and saying, 'Roger, your hair's falling out.'
"I was very self-conscious about it. I felt inadequate and embarrassed."
As his hair thinned down his centre parting, Roger adopted a comb over and started working out in the gym, hoping a toned body would detract from his baldness.
He said: "Because everyone at the gym was in to how they looked though, they were quick to point out my thinning hair.
"I worked hard on having a good body, but I still felt old before my time and shy with women."
Still, at 23, Roger met Claire and fell in love.
"She never said anything about my hair, probably as she didn't want to hurt my feelings," he added.
"But, at 24, I became so concerned about my hair loss, I started buying a hair strengthening product for £50 a bottle.
"I used it for three years and must have spent £1,800 on it – although it didn't work.
"I didn't tell anyone I was using it and felt like I was buying something off the top shelf.
"I do worry now that Asher might suffer from the same problem when he gets older, too, but, luckily, Seth and Theo have much thicker hair."
The more Roger, who owns three businesses, discussed having a transplant with Owen – who he remembers wearing baseball caps to cover bald patches – the more it made sense for him to have one, too.
So, they both booked into the Bella Vou Clinic in Tunbridge Wells, Kent – with Owen going first on March 11 and Roger a day later, for a pricier £6,000 procedure, giving a more natural look.
Both operations took four-and-a-half hours.
Single Owen, who is working temporarily as a contract cleaner, had strips of skin, with 2000 follicles attached, removed from the back of his head – leaving a small scar – and transplanted across the top, by a team of six, as his dad watched.
Owen said: "It didn't hurt. In fact, it was boring.
"I was fine with my dad watching. I just asked how much hair he would have as I didn't want him to have more hair than me!
When he first saw the results, Owen said he regretted it, but that soon wore off.
"I looked so ugly. I had a bruised, blue head," he recalled.
"Afterwards, I was impatient for my hair to grow, even though we'd both been told it would take 18 months to see the full effect.
"But, by June, I noticed a difference. My hair thickened and filled in the arches.
"And I went out for a family meal, for my 19th birthday, feeling a lot more confident."
While Roger was surprised by the sore appearance of Owen's head, after the op, it didn't put him off.
The next day a six-strong team transplanted 2,500 follicles for him, individually, rather than on strips of skin like Owen's.
He said: "I wanted to look like I was receding a little, so it didn't look fake.
"I was a bit worried, as I was used to how I looked.
"And, while the procedure wasn't painful, I was left with a slightly purple, swollen head."
At first, their scalps scabbed over, which was unsightly.
But, after three months, their first hairs sprouted.
Roger said: "I'm really happy with what I'm seeing now.
"Owen is far more confident about his appearance and I have to admit, I am, too."


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Portal:Mass surveillance
Mass surveillance is the intricate surveillance of an entire or a substantial fraction of a population in order to monitor that group of citizens. The surveillance is often carried out by local and federal governments or governmental organisations, such as organizations like the NSA and the FBI, but it may also be carried out by corporations (either on behalf of governments or at their own initiative). Depending on each nation's laws and judicial systems, the legality of and the permission required to engage in mass surveillance varies. It is the single most indicative distinguishing trait of totalitarian regimes. It is also often distinguished from targeted surveillance.
Mass surveillance has often been cited as necessary to fight terrorism, prevent crime and social unrest, protect national security, and control the population. Conversely, mass surveillance has equally often been criticized for violating privacy rights, limiting civil and political rights and freedoms, and being illegal under some legal or constitutional systems. Another criticism is that increasing mass surveillance could lead to the development of a surveillance state or an electronic police state where civil liberties are infringed or political dissent is undermined by COINTELPRO-like programs. Such a state could be referred to as a totalitarian state.
Selected article
Featured Picture
The official logo of NROL-39, an American reconnaissance satellite operated by the National Reconnaissance Office and launched in 2013. This logo drew criticism following the ongoing surveillance disclosures; satirist Jon Stewart described it as the government "owning the fact that they are getting nefarious" with "a giant octopus sucking the face off North America". According to the NRO, the logo's "octopus [is] a versatile, adaptable, and highly intelligent creature" from which America's enemies cannot hide.
Selected programme
ECHELON, originally a code-name, is now used in global media and in popular culture to describe a signals intelligence (SIGINT) collection and analysis network operated on behalf of the five signatory states to the UKUSA Security Agreement[2] (Australia, Canada, New Zealand, the United Kingdom, and the United States, referred to by a number of abbreviations, including AUSCANNZUKUS[2] and Five Eyes).[3][4][5] It has also been described as the only software system which controls the download and dissemination of the intercept of commercial satellite trunk communications.[6] It was created in the early 1960s to monitor the military and diplomatic communications of the Soviet Union and its Eastern Bloc allies during the Cold War, and was formally established in the year of 1971.[7][8]
By the end of the 20th century, the system referred to as "ECHELON" had evolved beyond its military/diplomatic origins, to also become "... a global system for the interception of private and commercial communications."[9]
The system has been reported in a number of public sources. One of the earliest reports to describe the programme, code-named "ECHELON", was Duncan Campbell's 1988 article, "Somebody's listening", published in the New Statesman.[10] The programme's capabilities and political implications were investigated by a committee of the European Parliament during 2000 and 2001 with a report published in 2001,[9] and by author James Bamford in his books on the National Security Agency of the United States.[6] The European Parliament stated in its report that the term ECHELON is used in a number of contexts, but that the evidence presented indicates that it was the name for a signals intelligence collection system. The report concludes that, on the basis of information presented, ECHELON was capable of interception and content inspection of telephone calls, fax, e-mail and other data traffic globally through the interception of communication bearers including satellite transmission, public switched telephone networks (which once carried most Internet traffic) and microwave links.[9]
Bamford describes the system as the software controlling the collection and distribution of civilian telecommunications traffic conveyed using communication satellites, with the collection being undertaken by ground stations located in the footprint of the downlink leg.
Did you know?
Did you know...
From new and recently improved content by Wikipedia's WikiProject Mass surveillance in recognition of The Day We Fight Back:
Links to related subjects
- Surveillance state
- Right to privacy
- WP:Mass surveillance
- RN on Mass surveillance
- Mass surveillance
- Edward Snowden
- Julian Assange
- Chelsea Manning
- PATRIOT Act
- George Orwell
- Big Brother (Nineteen Eighty-Four)
- ^ The Privacy Torts
- ^ a b
Given the 5 dialects that use the terms, UKUSA can be pronounced from "You-Q-SA" to "Oo-Coo-SA", AUSCANNZUKUS can be pronounced from "Oz-Can-Zuke-Us" to "Orse-Can-Zoo-Cuss".
- From Talk:UKUSA Agreement: Per documents officially released by both the Government Communications Headquarters and the National Security Agency, this agreement is referred to as the UKUSA Agreement. This name is subsequently used by media sources reporting on the story, as written in new references used for the article. The NSA press release provides a pronunciation guide, indicating that "UKUSA" should not be read as two separate entities. (The National Archives) (National Security Agency)
- ^ "UK 'biggest spy' among the Five Eyes". News Corp Australia. Retrieved 19 October 2013.
- ^ Google books – Echelon by John O'Neill
- ^ "AUSCANNZUKUS Information Portal". auscannzukus.net. Retrieved 1 February 2010.
- ^ a b Bamford, James; Body of Secrets, Anchor, ISBN 0-385-49908-6; 2002
- ^ "Q&A: What you need to know about Echelon". BBC. 29 May 2001.
- ^ Nabbali, Talitha; Perry, Mark (March 2004). "Going for the throat". Computer Law & Security Review. 20 (2): 84–97. doi:10.1016/S0267-3649(04)00018-4.
It wasn't until 1971 that the UKUSA allies began ECHELON
- ^ a b c Schmid, Gerhard (11 July 2001). "On the existence of a global system for the interception of private and commercial communications (ECHELON interception system), (2001/2098(INI))" (pdf – 194 pages). European Parliament: Temporary Committee on the ECHELON Interception System. Retrieved 5 January 2013.
- ^ Campbell, Duncan (12 August 1988). "Somebody's Listening". New Statesman. Archived from the original on 2007-01-03. Retrieved 16 September 2013.

subset: commoncrawl


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Learn techniques to raise the ceiling on how much you can earn per appointment. Introducing upsells, passive services, and treatment series can boost your profitability.
There are only so many hours in a day, and when you work in an appointment-based business, it's easy to feel like your income potential is limited by this fact. But what if you could increase your production within the service — along with the amount you can charge for it? How about offering add-on services that don't require your active involvement? This way earnings go up, but longer appointment times aren't needed.
Expanding profits without increasing service time begins during the client consultation, with a close analysis of the condition of the hands, feet, and nails. Let's say a client comes in for a manicure or pedicure and during the consultation she's found to have dry, rough hands or feet (not calluses). You can help this client with an upgrade that can be done without extending her original appointment time. First, during the consultation, talk to the client about what you're seeing on her hands or feet and what solutions are available: "Your hands are really rough, chafed, and dry. I suggest we add a scrub to gently remove the roughness, then a good hydrating product that can penetrate and soften your skin." Be certain to explain the charge for the upgrade.
If the client agrees, later in the service, you'll introduce the topic of home care products: "This lotion will allow your skin to hydrate at home, and then, at our next appointment, we will do a serious hydration treatment with paraffin mitts. You will begin seeing a dramatic change then."
The upgraded scrub and hydration treatment can be managed in the same time as her regular nail service. Begin by adding a scrub product at the beginning of the massage portion of the manicure or pedicure. The scrub is massaged on the skin to exfoliate it, then removed, if necessary. For the remainder of the massage, use a deep moisturizing product, such as one containing shea butter. With her rough, dry skin gone, the client pays for the upgrade, purchases a home care treatment lotion, and schedules an appointment for the following week for a specialty hydration treatment with paraffin mitts.
If a client comes in with aging, crepey skin, you can begin to treat the skin and get her ready for a series of anti-aging services starting with her next visit. Ask the client if she would like to see the crepiness reduced on her skin, then tell her how it is done over a series of treatments, with a start today. Explain that home care is important to get her hands ready for a treatment series.
The secret to adding the skin treatment without extending the service time is using a 15%-20% lactic- or glycolic-acid hand lotion. These solutions can be purchased from skin care companies; however, regulations vary from state to state as to the concentrations of liquid AHA products nail techs are allowed to work with. Instead I recommend purchasing lotions with a 15%- 20% concentration.
Then, the massage portion of the service is broken into two parts. The first three to five minutes of the massage is with the lactic- or glycolic-acid hand lotion. The rest of the massage is done with a lightening lotion (also available from skin care companies). Watch the hands closely during the massage with the glycolic- or lactic-acid lotion — any redness means it is time to stop and move on to the other product in the massage.
Upselling is not taking advantage of clients, as some technicians believe. Instead, it is meeting their needs and supporting their beauty — the very reasons they come into salons. Upsold services should account for 15% or more of a salon's gross revenue, but, sadly, that's rarely the case with nail departments. Improving the technician's consultation skills can change this. Client consultations that include upsells can grow salon and technician income dramatically.
To do effective client consultations, staff members must learn about common skin and nail conditions and be presented with the appropriate upsells for each condition. For example, is the skin dry? The technician can recommend a solution like exfoliation and paraffin mitts or heated mitts over a hydrating lotion. Home care treatments should be discussed as well.
Does the client chew her nails? A structured nail growth series should be recommended to this client. Are a client's nails weak and tearing off so they don't grow? A series of weekly nail treatments should be recommended to this client.
Before a technician can introduce upselling into the consultation, treatment products must be sourced and the products must be added to the treatment protocols. Staff members must be educated on the benefits of the active ingredients so they can share that with clients. The vital role home care products for effective treatment must also be taught so the technician can educate her clients.
Unlike active services that require the time and skill of a licensed professional, passive services don't require the tech to participate; instead, a piece of equipment performs the service. Passive services can be an upgrade to an active service or a standalone service. These are some examples:
LED light therapy for hands: This therapy is an anti-aging treatment and the services are sold as a series. The client is encouraged to come into the salon between manicures to sit for a 15-minute treatment, as well as adding a treatment before or after her regular manicure. I recommend the Hand Spa by Revitalight (www.revitalight.com) because clients must come into the salon to use it and a professional isn't needed to set things up or monitor the client.
These treatments have been proven to work for the skin on any area of the body, but must be performed often and with home care to be effective. A client can purchase several weeks of treatments, along with sunscreen. Sunscreen must be used during and after the treatment period or it will not be effective.
LED light therapy for feet: This treatment is basically the same as for hands, but a different light that's specially designed for the feet is used.
Hydrotherapy chair or bed: This equipment can be used as a passive massage treatment or to calm the nerves of clients when the technician is running late. It is often included in packages as "passive massage," and is sold by the minute as a standalone treatment, usually at $1-$2 per minute. Clients do not have to undress to use the bed or chair and those who love it will schedule it as a standalone treatment or before or after a manicure or pedicure. Many will just drop in, saying, "I need a massage!" and get on the bed or into the chair. The stress-relief quotient of this equipment is huge. You can learn more at www.hydromassage.com.
Salt room: A separate room can be set up as a salt therapy room where the client can come in with or without an appointment. The rooms usually have multiple lounge chairs. Breathing in the salt air promotes better breathing, healthier skin, sounder sleep, improved physical fitness and endurance, and overall wellness. Salt therapy salons are being set up around the country and are doing well.
Passive services require marketing. Fortunately, the equipment manufacturers usually provide training on how to promote the services. If you're considering purchasing passive therapy equipment, you should experience them personally first.
Selling treatment series can increase salon profits dramatically by raising the number of visits and the tickets per client. Series also require home care, so retail sales increase dramatically. In addition, they keep clients in the salon for maintenance after the series is completed.
Almost every service performed in a nail salon can be setup as a series of treatments. As with other upsells and add-ons, selling begins at the consultation. You must educate the client on what a series will do to improve the appearance of her hands, feet, or nails, and on the role home care plays in the improvement. All series should include photos before the first treatment and after the treatments conclude, all taken in similar lighting. Clients forget and the pictures will show them their improvement.
Examples of hand and foot series are:
Hydration therapy: Dry skin requires exfoliation, hydration, and maintenance. First, the skin is hydrated through a series of treatments, then maintenance services are a must.
Anti-aging treatments: Aging of the hands is inevitable, but appearance can be greatly improved with a series of anti-aging treatments, followed by maintenance. The treatments can also be enhanced and maintained through use of the LED light therapy.
Hyperpigmentation: This service manages discoloration on the hands/feet and requires a series of treatments with specialty products followed by maintenance with professional-grade home care products. These treatments can also be teamed with LED light therapy.
Callus control: The softening and reduction of calluses through a series of treatments and home care is welcomed by clients who have heavy calluses. After the calluses are controlled, maintenance is necessary.
Nail growth: This series includes a basic manicure, plus nail treatment and growth activities during the appointment, such as matrix massage on each nail and conditioning treatments for the nail plate.
Nail biters: Nail biters can benefit from a hard coating or bitter tasting top coat, as well as home care products like cuticle oil. These series tend to be somewhat open ended, continuing until the client is ready for routine services. (The sidewalls should be completely grown out and strong.)
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Established in 2007, the RB GROUP is a professional rally team made up from well experienced individuals with a strong mission & a deep passion that has become more than a daily need.
Our mission is to win & be leaders in our field .To do so, the job is not easy at all. Our strategy requires careful, long & hard hours of work, training & testing in order to achieve the wished goal.
In the RB GROUP, we share between us a strong will of improving ourselves continuously by practicing, analyzing our performance, & fine tune it so we can be more & more in the front rows.
After winning the 2017 FWD title in the speed test championship, we are looking forward to clinch more titles in the 2018.
Our 2018 Participation will include the rally championship including the 41th Rally of Lebanon.
Our website contains various sections that will enable you to get to know us more. Stay tuned to get the latest motorsport news of our team during rallies.


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Things You Won't Like About Paper Writing Web Sites and Things You Are Going To
The War Against Paper composing sites
You'll have to establish what you should compose in your essay. Even though some might phone essay composing an art, however if one understands the strategies and tricks and has now the capacity to master them, essay writing might never be this type of battle. The essay should be well-written and well balanced little bit of writing. Hence, to put an essay that is expressed a little value become set on that. The thing that is best about composing an informative essay may be the broad choice of subjects you can pick from. A person must learn how to write an elegant, clear and essay that is beneficial it is the easy percentage of our scholastic. To compose an essay that is great need to be familiar to kinds.
Informative message documents may be written on a number of subjects and so the extremely first is action is choosing the absolute most topic that is appropriate your paper. The paper may additionally poorly be organized pertaining to paragraphs and syntax. a customized written paper is certainly one that will be written exactly for your requirements. Regardless of fact that is simple composing a paper might appear become meticulous and problematic in begin, it truly is not really muddled when you the precise format and data flow to receive it finished. Whenever you get a study paper writing project, it is extremely typical to locate a study paper writing business which can help you build your research documents. Nonetheless, the genuine key to a thriving research paper is organization. an exceptional research paper writing business is a superb company that provides pupils with internet research paper assistance.
the significance of Paper Writing Websites
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Things you need to know About Paper Writing internet sites
if you should be a do-it-yourselfer, check out tips for composing internet content works well. Even though the written text could be thing that is potent images enable readers to rapidly grasp the principles presented in the paper, particularly into the systematic industries where a large amount of figures can be necessary in presenting information. Using the written word to educate or advertise is a technique that features existed for years and years.
You need to avoid plagiarizing the task inside an example paper and try to create your ideas that are own you completely see the test. It must be clear to aid facilitate the work of composing the study paper. The expression paper writing has to be completed having a step by step procedure and in advance work concerning the topic. OR interview your self just like you're already a writer that is successful. Our online essay article writers can be obtained throughout to ensure you've got all the thing you need at any time that is particular. Or you're fortunate, you are able to understand an individual who's a writer that is professional's willing to assist you. The time paper author should be aware firstly the right method of composing the paper.
Finally, you need to pick topics any particular one feels comfortable to create in in order to do justice for them. You will need to see the subjects where you are able to offer strategies that are useful responses towards the concerns that folks ask. This means you must strictly stay glued to the topic, plus don't try to reply to many concerns within the exact exact same essay. A number of the topics are so boring they will have no interest to start the research approach. It is vital to consult commonly topresume.com on the subject. Many the subjects used for composing process essays are connected with academics.
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There are numerous writing organizations providing aid in composing Spanish custom made research papers not all them offer high exceptional assistance. When you are working together with expert writing company, it is vital which you find business which well acts your passions. Dependable writing organizations supply the maximum quality Spanish tailor made research documents. Consequently, they should make certain that authors have the ability to create 100% initial documents. You do not be able to figure out respectable research paper writing organizations since many of them feel and look the very same.
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TP53 mutation is an independent marker of poor prognosis in patients with diffuse large B-cell lymphoma (DLBCL) treated with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (CHOP) therapy. However, its prognostic value in the rituximab immunochemotherapy era remains undefined. In the present study of a large cohort of DLBCL patients treated with rituximab plus CHOP (R-CHOP), we show that those with TP53 mutations had worse overall and progression-free survival compared with those without. Unlike earlier studies of patients treated with CHOP, TP53 mutation has predictive value for R-CHOP–treated patients with either the germinal center B-cell or activated B-cell DLBCL subtypes. Furthermore, we identified the loop-sheet-helix and L3 motifs in the DNA-binding domain to be the most critical structures for maintaining p53 function. In contrast, TP53 deletion and loss of heterozygosity did not confer worse survival. If gene mutation data are not available, immunohistochemical analysis showing > 50% cells expressing p53 protein is a useful surrogate and was able to stratify patients with significantly different prognoses. We conclude that assessment of TP53 mutation status is important for stratifying R-CHOP–treated patients into distinct prognostic subsets and has significant value in the design of future therapeutic strategies.
Diffuse large B-cell lymphoma (DLBCL) is the most common (30%-40%) and perhaps the most heterogeneous type of nonHodgkin lymphoma and has aggressive clinical features. Using gene-expression profiling (GEP), DLBCLs can be classified into 2 molecularly distinctive types: germinal center B-cell like (GCB) and activated B-cell like (ABC) that resemble the GEPs of normal germinal center B cells and mitogenically activated blood B cells, respectively.1 Despite this heterogeneity, DLBCL patients are usually treated with the same combination chemotherapy regimen, traditionally cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (CHOP), and in the past decade, rituximab plus CHOP (R-CHOP). R-CHOP has clearly improved the outcome of DLBCL patients. In a study of young patients, the 6-year event-free survival (EFS) was shown to be 74.3% and overall survival (OS) was 94.9%.2 In elderly patients, the rate of complete response (CR) has been shown to be 76%3; the 10-year progression-free survival (PFS) was shown to be 36.5% and the OS 43.5%.4 However, among patients with refractory disease or with early relapse, the CR rate was only 38%, and the 3-year EFS rate was only 31% after high-dose chemotherapy and autologous stem-cell transplantation.5 Therefore, the identification of prognostic biomarkers for high-risk DLBCL subgroups is essential for designing targeted chemotherapy.
The p53 (TP53) protein (393 aa) encoded by the TP53 gene is a crucial tumor suppressor that mediates cell-cycle arrest, DNA repair, apoptosis, senescence, and autophagy under cellular stress via transcription-dependent activity (TA) and transcription-independent activity (TIA) in the nucleus and cytoplasm, respectively.6,7 TP53 dysfunction is implicated in lymphomagenesis and disease progression,8,9 and normal function of TA and TIA of p53 is crucial for tumor suppression.10,11 Nonsynonymous TP53 mutations alter the p53 protein sequence and structure, disrupt function, and in many tumors are the most common mechanism that inactivates TP53. TP53 mutations could provide prognostic and predictive information to guide more targeted therapy for lymphoma patients.12
The DNA-binding domain (DBD) of p53 is the most important domain for its TA and TIA,13,14 so most TP53 mutations occur in the DBD encoded by exons 4-8 of the TP53 gene.15,16 In our previous study of TP53 mutations in 477 DLBCL patients treated with CHOP therapy,17 we detected exon 5-9 TP53 mutations in 21.4% of DLBCL patients, 66% of which occurred in the loop (L1)–sheet-helix (LSH) motif and 2 large loops L2 and L3, which are crucial for DNA binding.18,19 Functionally, TA of most p53 mutants have been characterized by a yeast-based functional assay20 and in some human and yeast lines, available in the International Agency for Research in Cancer (IARC) TP53 database (http://www.iarc.fr).15 Many p53 mutants (MUT-TP53) exhibit dominant-negative characteristics on wild-type p53 (WT-TP53) function by associating together as tetramers.
The prognostic significance of TP53 mutations has been inconsistent in several cancers, probably because of limitations of mutation detection methods, heterogeneity of TP53 mutations, inherent heterogeneity of disease, different therapies, diversity of p53 functions, and other mechanisms that inactivate the TP53 pathway.13,21,22 Our study and others have shown that TP53 mutation is an independent prognostic indicator of poor survival in DLBCL patients treated with CHOP chemotherapy.16,17,23⇓–25 In our earlier study using a different DLBCL patient cohort treated with CHOP therapy, TP53 mutations were correlated with worse OS, mostly attributable to the poor prognostic impact of TP53 mutations in the DBD. When we subgrouped patients into GCB- and non–GCB-DLBCL based on GEP data, the predictive value of TP53 mutations was restricted to patients with GCB-DLBCL.17
The anti-CD20 Ab rituximab combined with CHOP has significantly improved survival of DLBCL patients. Rituximab acts through 3 mechanisms: direct signaling, complement-mediated cytotoxicity, and Ab-dependent cellular cytotoxicity.26 Influx of external calcium (Ca2+) is essential for signaling-induced apoptosis mediated by the BCR and CD20.27,28 Therapeutic (high) doses of rituximab induce intracellular Ca2+ mobilization independently of CD20 and inhibit tumor cell growth.29 The effects of rituximab are attributed to inhibition of the BCR-signaling cascade,30 p38, NF-κB, ERK, and Akt and down-regulation of cytokine IL-10 and BCL-2.26 The TA and function of p53 might differ after rituximab addition as a result of different stresses induced by R-CHOP and CHOP because the p53 transcriptional program is stimulus and cell-type specific.13,31 The TIA of p53 can also be affected by the p53-independent signaling pathways induced by rituximab. Therefore, the prognostic value of TP53 mutation in DLBCL patients treated with R-CHOP needs to be evaluated.
To address the prognostic significance of TP53 mutations in DLBCL, in the present study, we analyzed exons 2-11 of TP53 in a large cohort of de novo DLBCL patients treated with R-CHOP and compared mutation status with clinical outcome.
The study cohort consisted of 506 de novo DLBCL patients treated with R-CHOP therapy in 29 medical centers selected based on the eligibility and exclusion criteria of histology, treatments, availability of clinical data, and biologic results. All patients were diagnosed according to the World Health Organization (WHO) criteria and reviewed by a group of hematopathologists (participating center pathologists were S.M.-M., M.A.P., M.B.M., A.T., and K.H.Y.). Patients with primary mediastinal large B-cell lymphoma, cutaneous or CNS DLBCL, HIV infection, or a history of low-grade B-cell lymphoma with transformation to DLBCL were excluded.32,33 Eligible patients were diagnosed between June 15, 1998 and October 8, 2008 (14 patients diagnosed in 1998-1999 were enrolled in the R-CHOP clinical trials). Most patients (n = 416) underwent 6-8 cycles of R-CHOP-21; others (n = 90) received R-CHOP–like regimens. In 182 patients, radiotherapy (25-50 Gy) followed R-CHOP and in 29 patients, hematopoietic stem cell transplantation followed R-CHOP (7 patients received both radiotherapy and transplantation). Treatment responses (CR, partial response, stable disease, and progressive disease) and follow-up were evaluated according to the recommended criteria.34,35 Thirty-eight percent (193 of 506) of the patients had died by the last follow-up. The median follow-up interval for the 313 censored patients was 59.73 months (range, 12-180.26). The present study, conducted in accordance with the Declaration of Helsinki, was approved as being of minimal or no risk or as exempt by the institutional review boards of all participating centers, and the overall study was approved by the institutional review board of the University of Texas MD Anderson Cancer Center.
TP53 mutations by resequencing microarray
Genomic DNAs extracted from formalin-fixed, paraffin-embedded (FFPE) tissues were used for TP53 exon sequencing. Sequencing of the coding sequence (exons 2-11) and splicing sites was performed using p53 AmpliChip (Roche Molecular Systems).36 For data analysis and classification, TP53 sequences were generated and compared with the TP53 reference sequence (NC_000017.10) in the GenBank database.
GEP and subtype classification
Total RNAs extracted from FFPE tissues were subjected to GEP using Affymetrix GeneChip Human Genome HG-U133 Plus Version 2.0 to classify 506 DLBCL patients into GCB or ABC subtypes, as described previously.33 Immunohistochemistry (IHC) based on staining of biomarkers including CD10, BCL-6, GCET-1, FOXP-1, and MUM-133 was performed on tissue microarrays to classify the 506 patients according to the Visco-Young algorithm with high concordance with GEP analysis, as described previously.33
FISH for TP53 gene (chromosome 17p13.1) deletion
Among the 506 DLBCL patients studied by GEP, 440 FFPE tissue sections were available for evaluation of chromosome 17p13.1 deletions by interphase FISH using a LSI TP53 Spectrum Orange Probe (Vysis) and methods described previously.16 A CEP 18 Spectrum Aqua Probe (Vysis) was used to evaluate simultaneously for chromosome 17 copy number. The ratio of TP53 signals (orange) to centromere 17 signals (green) was counted in 200 tumor cells. A ratio lower than 0.81 was considered to support the presence of a TP53 deletion. Because no patients had a ratio below 0.5, it was assumed that no homozygous deletions occurred.
IHC for p53
IHC staining was performed for all patients using a method described previously and the DO-7 mAb (DAKO).16 The percentage of positive tumor cells for each DLBCL patient in 5% increments was evaluated independently by a group of 6 pathologists in addition to each contributing center pathologist, and disagreements were resolved by joint review on a multihead microscope. Receiver operating characteristic (ROC) curve analysis was performed using Prism Version 5 software (GraphPad) to assess the discriminatory accuracy of p53 protein overexpression in predicting TP53 mutation status.
Statistical analysis
A comparison of clinical and laboratory features at the time of presentation between different DLBCL subgroups was carried out using the χ2 test and the Spearman rank correlation. OS was calculated from time of diagnosis to time of death from any cause. PFS was calculated from the time of diagnosis to the time of progression or death from any cause.35 Patients who remained alive or progression free were censored at the time of last follow-up. Multivariate analysis for survival of the study cohort was performed with SAS Version 9.3 software for Windows (SAS Institute) using the Cox proportional hazards regression model. OS and PFS curves of different groups were analyzed by Prism 5 using the Kaplan-Meier method and differences were compared using the log-rank (Mantel-Cox) test. All differences with P ≤ .05 were considered to be statistically significant.
Most TP53 mutations present in DLBCL are missense mutations in the DBD
Gene sequencing detected 133 TP53 gene mutations in 112 DLBCL patients, including 111 (21.9%) patients with nonsynonymous mutations or p53 mutants (MUT-TP53) and 17 patients with multiple (2-4) mutations. Mutation prevalence and patterns are shown in Figure 1A-B and Table 1. The 112 patients with 133 TP53 mutations included 92 with missense mutations (including 15 patients who carried 2 missense mutations); 14 with a nonsense mutation; 2 with a 2-bp deletion resulting in frameshift; 3 with a silent mutation; and 7 carrying a mutation in splicing sites. Missense mutations accounted for 81% of the total mutations present in this cohort.
The exon and codon distributions of these mutations are shown in Figure 1D and E. Exons 5-8 were most often mutated. A total of 117 (88.6%) mutations occurred in the DBD, including 25 (18.9%) in L3, 24 (18.2%) in the LSH motif, and 20 (15.2%) in L2. Codons 248, 273, 175, 176, and 213 of the p53 protein had the highest mutation frequency. Codons 248, 273, and 175 are the TP53 mutation hot spots found in most human cancers.
Most missense and nonsense mutations are loss-of-function mutations
Functional analysis of TP53 mutations using bioinformatics models.
Functional prediction of the 121 missense or nonsense mutations was computed by SIFT (http://sift.jcvi.org/) and AGVGD (http://agvgd.iarc.fr/) methods based on protein sequence homology. By SIFT, 108 (90%) missense or nonsense mutations were predicted to be deleterious (disrupt the p53 function), and 12 (10%) missense or nonsense mutations were predicted to be tolerable (neutral). By AGVGD, 99 (81.8%) missense mutations were predicted to be deleterious and 8 (6.6%) missense mutations were predicted to be neutral.
Yeast-based functional assay data in IARC database.
Effect of mutations on p53 transcription function can be retrieved from the International Agency for Research on Cancer (IARC) TP53 database (http://www.iarc.fr; TP53MutFunction2R15 dataset) that was established by a p53 transactivation activity assay in 8 yeast promoters (WAF1, MDM2, BAX, 14-3-3δ, p53AIP1, GADD45, NOXA, and p53R2) that harbor p53 response elements. Of 133 detected mutations, there were 99 mutation variants (some patients carried the same TP53 mutation variant), including 79 missense mutation variants, 9 nonsense mutations resulting in truncated protein, 6 splicing mutation variants, 2 frameshift mutation variants in exon 6 or 7, and 3 silent mutations. Among the 88 missense and nonsense mutation variants, 66 (75%) were nonfunctional, 16 (18.2%) partially functional, and only 4 (4.5%) functional, as experimentally measured by the yeast-based functional assay (Figure 1C). These data correspond to 94 (77.7%) nonfunctional, 18 (15%) partially functional, and 5 (4%) functional mutations among the 121 detected missense and nonsense mutations, with 4 missense or nonsense mutations uncharacterized. Splicing mutations and frameshift mutations result in more alterations of protein sequence than missense mutations and therefore potentially have a more deleterious effect on p53 function. Altogether, more than 90% of mutations result in loss of p53 function. Most mutations at hot spots, including R248W, R248Q, R273C, R273H, R175H, C176R, R213X, and R213Q, are nonfunctional, whereas C176F is partially functional, and R175C is functional. Furthermore, according to the IARC TP53MutFunction1R15 dataset, 32 mutants detected have a dominant-negative effect on p53 transcription function, and 3 mutants have lost the ability to form tetramers.
Functional data for a limited number of missense and nonsense mutants regarding TIA are available in the IARC TP53MutFunction1R15 dataset. R175H, Y234C, G245S, R248Q, R248W, R273H, C277Y (partial), R280K, and R337C (partial) have lost the ability to induce apoptosis in human cells, whereas S121F, R175C, R181C, and S240R are functional or partially functional regarding transcriptional activities and have conserved apoptosis function. Altogether, either by bioinformatic computations or by laboratory-based evaluation, approximately 90% of TP53 mutations detected in this study cohort were accompanied by a loss of p53 function.
Clinical features of the cohort
The clinical features of the WT-TP53 and MUT-TP53 groups are compared and summarized in Table 2. There were no significant differences between these 2 groups regarding age, sex, stage, B symptoms, number of extranodal sites, performance status, tumor size, or International Prognostic Index (IPI). Serum lactate dehydrogenase level (P = .05), tumor size (P = .02), and therapeutic response (P < .0001) were the only variables that were significantly different between these 2 groups.
Most patients were able to be classified into GCB- and ABC-DLBCL subtypes according to the GEP data (n = 441), supplemented by IHC assessment (n = 499). Of these, 258 patients were determined to be the GCB subtype, including 190 patients with WT-TP53 and 68 patients with MUT-TP53; 241 patients were determined to be the ABC subtype, including 198 patients with WT-TP53 and 43 patients with MUT-TP53. Serum lactate dehydrogenase (P = .005), tumor size (P = .013), and therapeutic response (P = .0009) differed significantly in patients with WT-TP53 versus MUT-TP53 in the GCB subtype, whereas only therapeutic response differed significantly in patients with WT-TP53 versus MUT-TP53 (P = .0113) in the ABC subtype.
Impact of TP53 mutations on survival in DLBCL patients treated with R-CHOP
TP53 mutations predict poor survival in all DLBCL patients.
Our results show that TP53 mutation status is a significant prognostic factor that stratifies DLBCL patients treated with R-CHOP. DLBCL patients with WT-TP53 had significantly better OS and PFS compared with DLBCL with MUT-TP53 (P = .0005 and P = .0004, respectively; Figure 2A-B). The median OS of 395 patients with WT-TP53 DLBCL was 94.49 months, in contrast to 52.90 months for 111 patients with MUT-TP53 (hazard ratio [HR] = 0.5320; 95% confidence interval [CI], 0.37-0.76). Patients with MUT-TP53 were associated with a significantly higher, 1.9 times (95% CI, 1.32-2.68) hazard for OS (poorer survival) compared with patients with WT-TP53. The 5-year OS was 65.9% for patients with WT-TP53 versus 47.8% for those with MUT-TP53 DLBCL. The median PFS for patients with WT-TP53 DLBCL was 93.14 months versus 51.95 months for patients with MUT-TP53 DLBCL (HR = 0.5370; 95% CI, 0.38-0.76). The 5-year PFS was 63.5% for DLBCL patients with WT-TP53 versus 46.3% with MUT-TP53.
TP53 mutations predict poor survival in both the GCB- and ABC-DLBCL subtypes.
GCB-DLBCL patients had significantly better survival than ABC-DLBCL patients (supplemental Figure 1A-B, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). GCB-DLBCL patients with WT-TP53 had better OS (P = .0024; HR = 0.46; 95% CI, 0.28-0.76) and PFS (P = .0013; HR = 0.4483; 95% CI, 0.28-0.73) than GCB-DLBCL patients with MUT-TP53 (Figure 2C-D). Similarly, ABC-DLBCL patients with WT-TP53 had better survival than ABC-DLBCL patients with MUT-TP53 (P = .0112, HR = 0.5218, and 95% CI, 0.32-0.86 for OS; P = .0259, HR = 0.5665, and 95% CI, 0.34-0.93 for PFS; Figure 2E-F).
Multivariate analysis.
Multivariate analysis showed that an IPI score of > 2, TP53 mutation, ABC subtype, and B symptoms were the only prognostic factors that independently predicted worse OS and PFS in DLBCL patients treated with R-CHOP (Table 3). TP53 mutation is an important prognostic marker second to an IPI score of > 2. Patients with TP53 mutation had a 1.95 times hazard for OS (P = .0004) and a 1.84 times hazard (P = .001) for PFS compared with patients without TP53 mutation.
TP53 mutations in the LSH, L2, and L3 motifs predict poor survival in DLBCL patients.
Because most (105 of 111; 95%) MUT-TP53 DLBCL patients carried mutations in the DBD, survival curves for DLBCL patients with DBD TP53 mutations versus those with WT-TP53 closely mirrored the overall MUT-TP53 versus WT-TP53 data (figures not shown). Mutations in the 3 important motifs for DNA binding, LSH, L2, and L3, were all significantly associated with worse survival (Figure 3A-F). Mutations in LSH (including the codon 273 hot spot) and L3 (including the codon 248 hot spot) were particularly associated with decreased survival: for LSH, a median OS of 26.76 months and PFS of 27.64 months, and for L3, a median OS of 30.64 months and PFS of 23.23 months. The median OS and PFS for L2 mutations (52.90 and 48.99 months, respectively) were similar to those of DLBCL patients with MUT-TP53 overall.
Differentially expressed genes in MUT-TP53 and WT-TP53 patients
To identify the genes responsible for the different clinical outcomes of DLBCL patients with WT-TP53 versus MUT-TP53 and the different therapeutic response in DLBCL patients with GCB versus ABC subtypes, we profiled and compared the GEPs of WT-TP53 and MUT-TP53 DLBCLs (Figure 4A-B). Twenty probe sets were significantly differentially expressed between WT-TP53 and MUT-TP53 DLBCL patients. Genes up-regulated in WT-TP53 DLBCL included: DOK7, DHRS9, HLA-DPB2, BATF3, RPS27L, and DUSP2; genes down-regulated included: PEG10, CDKN2A, CTAG1A/CTAG1B, TMEM97, NDC80, LOC96610, KIAA0020, and DKC1.
The impact of p53 status on GEP was different in GCB- and ABC-DLBCL. In GCB-DLBCL (Figure 4B), 40 probe sets were differentially expressed between MUT-TP53 and WT-TP53. Genes up-regulated in WT-TP53 included: HOPX, DHRS9, LOC100510485, CACNA1D, BATF3, DNER, SOCS1, CAMTA1, LOC729121, and MPP4; genes down-regulated included KIAA0101, PEG10, TMEM97, GPR180, TAF4B, CDKN2A, FAM72A, FAM72B, FAM72C, FAM72D, MCM4, E2F1, ATAD2, SLC16A1, NDC80, FIGNL1, WDHD1, NCAPG2, DKC1, SQLE, NDUFA1, CDK4, EIF4G1, DUS4L, TFAM, PA2G4, PA2G4P4, and APLP2. In contrast, in ABC-DLBCL, only 1 gene (PSMD10) was significantly down-regulated in the WT-TP53 compared with the MUT-TP53 patients. The functions and subcellular locations of these genes (http://www.uniprot.org) are listed in supplemental Table 1.
Association of TP53 mutations and LOH
TP53 single-allele mutations are frequently followed by loss of heterozygosity (LOH), further promoting tumor development and disease progression.22,37 In the present study, we observed a weak association between TP53 mutations detected by p53 AmpliChip and TP53 deletions detected by FISH. Ninety-four MUT-TP53 and 346 WT-TP53 DLBCL patients were assessed by FISH (17 MUT-TP53 and 49 WT-TP53 patients had no tissue available or FISH failed). Overall, 12.3% of DLBCLs had allelic deletion, with a lower frequency than mutation (21.9%). In the MUT-TP53 group, there were 16 (17.0%) patients with TP53 allelic deletion. In comparison, 38 (11.0%) patients in the WT-TP53 group had TP53 allelic deletion. Therefore, TP53 deletion (LOH) was more common in DLBCL with MUT-TP53; however, the frequency difference did not reach statistical significance (P = .1136). Conversely, the TP53 mutation frequency was higher in DLBCLs with allelic deletions (29.6%) than in tumors without deletions (20.5%), but this was also not statistically significant (P = .1253). These results suggest a weak association between allelic deletion and TP53 mutations.
Others have reported that complete inactivation of TP53 by concomitant TP53 mutations and LOH is present most often in high-grade lymphomas and might be associated with high-grade transformation or disease progression.38 In the present DLBCL study cohort, a significantly higher frequency (P = .025) of allelic deletion was present in DLBCL patients with a high proliferation index (Ki-67 protein ≥ 70%; high Ki-67 = 7.4% vs low Ki-67 = 14.9%). TP53 mutations also occurred significantly more frequently (P = .0235) in DLBCLs with a high Ki-67 index (high = 25.7% vs low = 16.8%). In addition, 15 of 16 DLBCLs with LOH concurrent with TP53 mutation had a high Ki-67 index. These data suggest that TP53 mutations and LOH are associated with a high Ki-67 index.
Impact of TP53 allelic deletion on patient survival.
Our results show that TP53 allelic deletion was not significantly associated with decreased OS or PFS (Figure 5A-B). The impact of allelic deletion might have been attenuated by the poor survival potential of DLBCL patients with MUT-TP53 with or without allelic deletion. Therefore, we further examined the impact of TP53 allelic deletion after stratifying patients into the WT-TP53 and MUT-TP53 DLBCL subgroups. In DLBCL patients with either WT-TP53 or MUT-TP53, allelic deletion was not correlated with survival (Figure 5C-F.), although allelic deletion tended to be correlated with decreased survival in DLBCL patients with WT-TP53.
Because LOH and TP53 mutations are significantly associated with high Ki-67, we assessed the impact of LOH and TP53 mutations on survival in patients with a high or low Ki-67 index. TP53 mutations, but not allelic deletion, affected survival significantly in DLBCL patients with highly proliferative tumors (Ki-67 ≥ 70%; Figure 6A-D).
p53 overexpression by IHC in tissue sections.
The p53 protein expression level was determined in 483 DLBCL patients using IHC. Different cutoffs of positive cells yielded different sensitivity and specificity by ROC analysis (supplemental Table 2). Cutoffs between 10% and 60% stratified DLBCL patients into 2 groups with significantly different OS and PFS (P < .05; supplemental Table 2). However, p53 overexpression determined by IHC using cutoffs of 10%-40% is less discriminative than TP53 mutation status to stratify DLBCL patients into groups with different survival. Although a 30% cutoff of p53 expression by IHC had the biggest Youden index, a cutoff of 50% had a higher specificity, highest HR, and best P value in patient stratification. Therefore, we believe that > 50% is the best p53 expression cutoff with which to stratify DLBCL patients in diagnostic practice if sequencing of TP53 is not available.
In the present study, we found a TP53 mutation prevalence of 21.9% in DLBCL patients, and the mutation patterns were similar to patterns in some other cancers. Most TP53 mutations were missense mutations, suggesting the importance of maintaining p53 structure in tumor suppression. In addition, most mutations occurred in the DBD, with hot spots at codons 248 (in L3), 273 (in LSH), and 175 and 176 (in L2), indicating that these motifs are critical for p53 tumor-suppression function. Bioinformatics tools based on alignments of TP53 sequences during evolution predicted that 90% of these missense mutations disrupt p53 function. Functional assays in human or yeast cells confirmed that most of these mutants lose transcriptional function. Impairment of transcription-independent apoptosis function by TP53 mutations has also been reported previously.39⇓–41 The mutants R273H, L194F, R175H, and E285K have a low ability to translocate to mitochondria and induce apoptosis under stress conditions42; therefore, loss of p53 TA or TIA could be a driver for lymphomagenesis.
In this large cohort of de novo DLBCL patients treated with R-CHOP, we show that TP53 mutations, especially mutations in the L3 and LSH motifs, are correlated with disease progression and poor survival (Figures 2 and 3). To eliminate the possible impact of radiotherapy and transplantation on survival, we also performed survival analysis for patients who received standard R-CHOP therapy only and found that patients with WT-TP53 had significantly better OS (P = .0041) and PFS (P = .0084). Therefore, despite the addition of rituximab to CHOP, TP53 mutation is an independent prognostic factor that predicts poor survival in patients with DLBCL. This observation holds true in patients with the GCB or ABC subtypes of DLBCL (Figure 2C-F) and further supports the crucial role of p53 in death of tumor cells and tumor suppression. Nonetheless, the impact of TP53 mutation on survival of GCB-DLBCL patients is more pronounced than for ABC-DLBCL patients (Figure 2C-F). In fact, TP53 mutation status was not an independent prognostic factor in patients with ABC-DLBCL treated with CHOP.17 The distinctive molecular programs harbored by GCB- and ABC-DLBCL43 might explain the differences in treatment response and impact of TP53 mutations. Perhaps somatic hypermutation (SHM) in GCB-DLBCL under chemotherapy may trigger rapid apoptosis in WT-TP53 patients, whereas accumulation of more oncogenic events in MUT-TP53 makes these tumors resistant to apoptosis. In contrast, the SHM machinery is inactivated in ABC-DLBCL, which instead expresses a pre-plasma cell program. NF-κB and BCR signaling pathways are activated in ABC-DLBCL,43,44 causing resistance to apoptosis and chemotherapy, negatively affecting the survival of ABC-DLBCL patients with WT-TP53, and minimizing the prognostic value of MUT-TP53. Therefore, with CHOP treatment, TP53 status is an independent predictor of treatment response only in the GCB subtype. However, with R-CHOP therapy, rituximab inhibits the NF-κB and BCR signaling pathways, and ABC-DLBCL patients with MUT-TP53 have significantly worse survival than those with WT-TP53.
In addition, similar to CHOP regimen, ABC-DLBCL patients treated with R-CHOP have significantly inferior OS and PFS compared with GCB-DLBCL patients both in the entire DLBCL cohort (supplemental Figure 1A-B) and in the WT-TP53 subcohort (supplemental Figure 1C-D). Our multivariate analysis also suggested that ABC subtype independently predicted worse survival (Table 3). These data are in agreement with a recent study.45 Most of the clinical characteristics contributing to IPI score and IPI score itself were significantly different between GCB-DLBCL and ABC-DLBCL patients (supplemental Table 3) in overall DLBCL and DLBCL with WT-TP53, suggesting that molecularly distinctive types can be clinically reflected on IPI parameters. In DLBCL patients with MUT-TP53, IPI score was not significantly different between the GCB and ABC subtypes (supplemental Table 3); correspondingly, OS and PFS were not significantly different (supplemental Figure 1E-F). This might be because of the small cohort of DLBCL patients with MUT-TP53, but also might reflect the dominant adverse impact of p53 mutations on survival in both the GCB and ABC subtypes.
We also performed GEP in DLBCL patients with WT-TP53 and MUT-TP53, as well as according to GCB- and ABC-DLBCL subtypes (Figure 4). One critical feature between WT-TP53 and MUT-TP53 DLBCLs is that certain genes were differentially expressed in GCB-DLBCL, but only 1 gene (PSMD10, a negative regulator of RB1 and p53) was significantly down-regulated in ABC-DLBCL with WT-TP53. These results imply either absence or heterogeneity of the p53 transcription activities in ABC-DLBCL and probably indicate that p53 is less activated in ABC- compared with GCB-DLBCL, perhaps because of decreased activity of SHM. Similar phenomena were observed previously in DLBCL patients treated with CHOP.16,17 However, ABC-DLBCL patients with WT-TP53 still had better survival compared with patients with MUT-TP53 (Figure 2E-F), suggesting that transcription-independent p53 function is mainly responsible for the better survival of ABC-DLBCL patients with WT-TP53 treated with R-CHOP.
The GEP differences between WT-TP53 and MUT-TP53 DLBCLs (Figure 4) could be explained as loss or altered p53 function in MUT-TP53. From this perspective, loss of tumor-suppressor function of MUT-TP53 includes transactivation of several genes involved in signaling and transcription, including negative regulators of ERK, JNK, and cytokine signaling pathways; activators of the NOTCH1 pathway; and transrepression of several genes involved in antiapoptosis, cell-cycle progression, biosynthesis, proliferation, transcriptional activation, and G-protein signaling. Conversely, the difference in gene expression between the WT-TP53 and MUT-TP53 DLBCL patients may result from gain-of-function of some MUT-TP53 mutants. The tumor-suppressor gene CDKN2A (encoding p16INK4A and p14ARF) was down-regulated in WT-TP53 DLBCL patients (Figure 4A-B). p16INK4A can cause cell-cycle arrest by inhibiting CDK4; p14ARF sequesters the p53-negative regulator MDM2, thereby enhancing p53 level and activity, or induces cell-cycle arrest and apoptosis by p53-independent pathways. p14ARF also inhibits the function of BCL-6,46 which is expressed in B-cell germinal centers and activated in a subset of lymphomas.44 The repression of CDKN2A by p53 has also been reported in a cell line study,47 and might provide negative feedback to the TP53 pathway.48 The known p53 targets (eg, MDM2, BAX, and p21) did not appear to be differentially expressed (P = insignificant), reflecting the biologic heterogeneity of DLBCLs.
The prognostic value of p53 overexpression as a surrogate for TP53 mutations has been studied by others, but with inconsistent results. We used an IHC method to detect p53 overexpression and compared it with TP53 mutation status. We evaluated different cutoffs of p53 expression by ROC analysis and found that most cutoffs were able to stratify patients with significantly different OS and PFS. However, most stratification using IHC appears to be less discriminating than TP53 mutation status. In the present study, a > 50% cutoff was determined to be the best for diagnostic practice if gene-sequencing data are not available.
In contrast to TP53 mutations, we found that TP53 allelic deletion, which also may inactivate the TP53 pathway, had no significant impact on patient survival, either overall or within the WT-TP53 or MUT-TP53 subsets (Figure 5C-F). In fact, DLBCL patients with both LOH and MUT-TP53 had a slightly better survival than DLBCL patients with MUT-TP53 alone (Figure 5E-F). The higher frequency of TP53 mutations than allelic deletions in our cohort may imply that LOH occurred after TP53 mutation, which drives lymphomagenesis. LOH seemingly added no further selective growth advantage for tumor cells that carried TP53 mutation (Figure 5E-F), which probably reflects the dominant-negative effect exerted by MUT-TP53 on WT-TP53 expressed from the other intact allele and gain-of-function by MUT-TP53 resulting in a greater decrease in survival than loss-of-function by LOH. Alternatively, the slightly worse survival of MUT-TP53 patients (Figure 5E-F) could be caused by TP53 mutations present in the other allele. The AmpliChip we used for mutation detection cannot distinguish between homozygous and heterozygous mutations, but 13 of 78 MUT-TP53 patients without LOH carried 2 or 3 different TP53 mutations. The comparably weaker growth advantage provided by LOH compared with TP53 mutation explains the low percentage of allelic deletions (17%) in MUT-TP53 DLBCLs and also the absence of statistically significant association between TP53 mutations and LOH in this cohort. However, concomitant LOH and TP53 mutation were almost always in patients with a high proliferation index (Ki-67 ≥ 70%), suggesting an association of LOH with disease progression. Nonetheless, our results (Figure 6A-D) suggest that TP53 mutations and not LOH drive disease progression.
In conclusion, in the present study, we identified TP53 mutation patterns in a large cohort of patients with de novo DLBCL who were treated with R-CHOP and confirmed that TP53 mutation status is a valuable prognostic biomarker. TP53 mutations, especially the ones in the LSH, L3, and L2 motifs of the DBD, were significantly correlated with worse survival in patients with either GCB- or ABC-DLBCL. Therefore, therapeutic approaches targeting the inactivated TP53 pathway may further improve clinical outcomes of patients with DLBCL treated with R-CHOP.49,50
Contribution: Z.Y.X.-M., L.W., and K.H.Y. designed and conducted the research, performed and supported the statistical analysis, and wrote the manuscript; Z.Y.X.-M., L.W., C.V., Y.C.T., A.T., W.L., S.M.-M., K.D., A.C., A.O., Y.Z., G.B., K.L.R., E.D.H., X.F.Z., W.W.L.C., X.Z., J.H.v.K., Q.H., J.H., W.A., M.P., A.J.M.F., F.Z., J.N.W., W.X., J.L., R.S.G., Y.L, M.A.P., M.B.M., R.N.M., L.V.A., L.J.M., and K.H.Y. contributed vital new reagents, resources, and analytical tools under approval by the institutional review boards and the material transfer agreement; Z.Y.X.-M., C.V., A.T., S.M.-M., K.D., A.C., A.O., Y.Z., G.B., K.L.R., E.D.H., X.F.Z., W.W.L.C., X.Z., J.H.v.K., Q.H., J.H., W.A., M.P., A.J.M.F., F.Z., J.N.W., R.S.G., M.A.P., M.B.M., R.N.M., L.V.A., L.J.M., and K.H.Y. collected clinical and follow-up data under approval by the institutional review boards and the material transfer agreement; Z.Y.X.-M., and K.H.Y. analyzed the data; and all authors contributed vital strategies, participated in discussions, and provided scientific input.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
The DLBCL Rituximab-CHOP Consortium Program has the principal investigation center at The University of Texas MD Anderson Cancer Center in Houston, TX, and includes 29 participating centers. A material transfer agreement was established and approved by each of the participating centers of the DLBCL Rituximab-CHOP Consortium Program. For a complete list of DLBCL Rituximab-CHOP Consortium Program participating centers, please see the supplemental Appendix.
Correspondence: Ken H. Young, MD, PhD, The University of Texas MD Anderson Cancer Center, Department of Hematopathology, 1515 Holcombe Blvd, Houston, TX 77030-4009; e-mail: khyoung{at}mdanderson.org.
The authors thank their consortium program team of pathologists, hematologists, and clinicians; each of the contributing center principal physicians; their patients; and former and current hematopathology and hematology/oncology fellows for their support.
Z.Y.X.-M. is a recipient of the Shannon Timmins Leukemia Fellowship Award at The University of Texas MD Anderson Cancer Center. A.T. is a recipient of the Stiftung zur Krebsbekaempfung Zurich Grant 269 award. K.H.Y is supported by The University of Texas MD Anderson Cancer Center Institutional Research and Development Fund, Institutional Research Grant Award, MD Anderson Cancer Center SPORE Research Development Program Award, Gundersen Lutheran Medical Foundation Award, MD Anderson Cancer Center Collaborative Funds with Roche, HTG Molecular and Daiichi Sankyo, and the Forward Lymphoma Fund. This study was also partially supported by the National Cancer Institute/National Institutes of Health (R01CA138688, 1RC1CA146299, and P50CA142509).
There is an Inside Blood commentary on this article in this issue.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
- Submitted May 31, 2012.
- Accepted August 18, 2012.
- © 2012 by The American Society of Hematology


text:
International 5.5 Regatta 2018
By Jennifer Harker
Special weather advisories issued by Environment Canada for an afternoon of high winds (80 km/hr) plus squalls and rain forced the cancellation of races on the first day of the North American Fall Regatta on Friday in Midland, Ontario.
Two boats headed out for some last minute tuning in the morning before the predicted squalls including USA 75 Pride, under the new ownership of Joe Strelow of Ann Arbor, Michigan. Pride was designed by Texan Ernie Fay in 1962 and quickly rose through the ranks of racing. Attracted to her impressive race pedigree, Strelow bought the boat a few short months ago on July 22 with plans to race her this fall.
He quickly found she was in need of some focused TLC and major rejuvenation.
Told it was impossible to have her ready for September Strelow set out to prove the naysayers wrong. There were definitely low moments. "A boat is not a boat if it doesn't float," he said, remembering his dismay at discovering several major holes and spreading rot. "Rot, mixed with regret and despair," he said ruefully. However patience, persistence and passion paid off.
Strelow brought a regenerated Pride to Midland in mid-September. Thanks to the knowledge, expertise, guidance and spare parts of John Lister, Dean Harker and Matt Thurley her bare decks soon sprouted all the necessary hardware and fittings and Pride was race ready.
It is a perfect example of the teamwork and dedication of the North American 5.5 organization. Although racing 5.5s – and winning – is the end goal, they put equal effort into restoring and rejuvenating the fleet while building camaraderie and the social aspect of the group at the same time.
With wild winds predicted for Friday, the 5.5s stayed at the dock, but some skippers and crew decided to get back to the basics and headed out in Optis instead, joking that they needed some remedial racing lessons. Taking advantage of a weather window and the protected inner harbor eight boats enjoyed some good natured fun. Proving yet again, the worst day on the water is still better than the best day in the office.
John Lister USA 51 Purple Haze
Hugh Loughborough CAN 22 Nantoria
Don Cameron CAN 79 O'Kelly
Joe Lambert USA 102 Aeolus II
Matt Thurley USA 75 Pride
Dan Rossi USA 33 Savage
Dean Harker CAN 30 SummerIn Saturday's first race Pride took the favoured end and jumped out to an early lead. Summer was on her heels with Aeolus close behind. The race would prove to be exciting to watch with the fleet continually jockeying for position and it was anyone's race to take. Pride rounded the first and second marks well ahead but Purple Haze was coming fast after taking the neglected north side when the rest of the fleet stuck to the south. The strategy paid off and Purple Haze would take the race handily but the race committee had to be sharp as the next five boats crossed en masse with about 10 seconds separating second from sixth place. Shifty winds kicked up keeping crews on their toes for the second race that saw Summer jump out to an early lead. Spinnaker-proficiency was the key to victory as Purple Haze and Pride battled for position. Aeolus had a beautifully smooth set around the mark and pushed into third place. By the second mark Nantoria was firmly in first and never relinquished her position. Race committee opted for a shortened course due to the shifting winds and Nantoria cruised to victory, followed by Purple Haze and Pride. While the course was reset Savage joined the fray. Owned by Dan Rossi, she is another work in progress and will need more work before she is in top form. Savage showed her form early with her crew working her into a fifth place finish. Saturday would finish with Purple Haze registering four firsts and a second, followed by Nantoria with a first, two seconds and two thirds. O'Kelly finished the day in third overall with two seconds, two thirds and a fourth. It seemed like Mother Nature had blown out her energy and Sunday offered up light winds, keeping the racers to two just courses. Check https://www.sailwave.com/results/MBSC-2018-5-5%20Meter%20NA%20fall.htm for full results from the weekend.


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Arts Peer Assessors2 recent views 270 total viewsPeer Assessors assess applications against criteria and moderate with consideration of: available funding; balance across art forms; geographic spread; target groups and...
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You might not be, but I haven't seen very intelligent arguments from anyone proclaiming themselves to be a socialist
but, as far as Stalin stuff goes, I'm 4PT about it
usually not
(though libertarians are generally not better)
Yeah I guess you could pull the "it's not real libertarianism" thing out
As a counterpart to "it's not real socialism"
yeah, the no-true-Scotsman args have to be substantiated at least
otherwise, moving the goalposts like that is faggy
(or Pharisaic)
(but I repeat myself)
NSDAP, though, didn't use the word *socialist* for nothing
National Socialism is not simply national capitalism
though I do think it didn't go far enough
so it failed to whack exploiters who were not Jewish
failed to catch many of the biggest Jews who did deserve to die
and killed some Jews who really posed no threat
I don't think you're gonna find a lot of people here who support government control in our private lives
Post your political compass
haha, my political compass is red zone
Oh god
at base, I'm pretty Confucian
but for the nation to actually secure itself against foreign interference
If you're not screaming about revolution and you don't have hammer and sickles in your name, I don't really mind
I do think a lot of 'left'-sounding stuff has to be done
I probably don't agree with you but I don't mind you
revolution sometimes is necesssary, sometimes not
depends on the circumstances
This conversation is all over the place
@Platz is this too hammer-and-sickle for you? https://twitter.com/LueYee/status/774495223963611136
sorry, @Hernán Cortés, I contain multitudes
Would you agree or disagree that hierarchy is essential to civilization?
Personally I support your freedom to converse in a reasonable manner here, but I don't represent the people in charge
of c we need hierarchy
I even think China needs emperors back, but in a diff. way
I also think that's compatible with a lowercase national socialism
Largest Non-Nuke bomb is US arsenal.
without some of the stupid stuff Marx said
Would you agree or disagree that capitalism is the best possible system we can currently implement, not because it aims to maximize equality, but because it aims to maximize efficiency?
I disagree with the assertion that capitalism is the most efficient for attaining a functioning (and therefore just and secure) commonwealth


text:
Olafur Eliasson's Little Sun Has Big Plans for Bringing Light to All
Over a billion people don't have access to electricity, but an ambitious project is fusing design, art and technology to help fill the darkness.
Photo via Thabo Metsing
According to the International Energy Agency, in the year 2016 somewhere in the ballpark of 1.2 billion people, or 16 percent of the world's population, did not have access to electricity. This means no internet access, no television, and not being able turn on something as basic as an electric lamp to light up their home. Almost all of these people live in rural areas in sub-saharan Africa or Asia. There is an obvious link between access to energy sources and economic development, but the availability (and lack thereof) of electricity, and electric lighting, has major implications for a number of social issues like gender equality, health care and education.
This lack of access to something that is such a basic component of modern life is exactly what the artist Olafur Eliasson and engineer Frederik Ottesen are working to address. in 2012, they launched Little Sun, an ambitious, socially conscious business endeavor that creates solar powered products with the aim to bring electric light and energy to communities literally left in the dark.
Born in Denmark in the late 60s to Icelandic parents, and now primarily working out of his studio in Berlin, Eliasson seems to go big with everything he does. He is one of the most prolific contemporary artists and his large scale and immersive works like the The Weather Project occupy some of the most important spaces in the art world. He is also a professor at the Berlin University of the Arts, where he founded the aptly named Institute for Spatial Experiments. His work is not confined to any one medium, or only to be found in museums.
This familiarity with big, larger than life art projects make Eliasson a dynamic player when it comes to addressing the world's electricity and light deficit, as does his experience designing public installations powerfully harnessing natural light and solar energy. What he is trying to accomplish with Little Sun is no mere art project. The company is currently in its fifth year, and is preparing to launch a new product, the Little Sun Diamond, their pocket-sized solar-powered lamp, this fall.
VICE Impact caught up with Eliason in late August by phone as he took a break from bicycling home from his studio in Berlin.
VICE Impact: Your work often takes large-scale formats. Do you see that inspiration, that experience, influencing a lot of the art that you're most proud of?
Olafur Eliasson: I have always been interested in, and inspired by, nature and natural phenomenas, by landscapes and by light. For me, natural phenomena created an opportunity to take a closer look on the sensitivity that society had cultivated. Nature is meant to be a space in which we can have very different experiences. I'm very interested in natural phenomenons as a kind of democratic exercise.
How did you come to be a part of Little Sun?
The so-called art world has its elitist limitation. It's not necessarily the tool with which one can reach out into every part of the world. While traveling in East Africa I saw how children were using these lamps made out of glass bottles with a cotton wick through the top of the bottle, which was on fire. And I said to a child, "My god, you have a Molotov Cocktail on your table! What will you do if it falls over?" And the child said, "Yes, but I need to do my homework. I have it because I need light."
This was like an epiphany for me. I was completely naive. I thought it would be absolutely easy to actually replace it with something cheaper, sustainable, healthier, and great for doing your homework. A year later Frederik Ottesen and I founded Little Sun. What I knew from art is that things can be emotionally activating. Then Frederik said let's get high quality components for a low price.
That was exactly five years ago when the price of solar panels went down by two or three hundred percent. That meant suddenly you could compete with the oil industry. That's how we kind of got involved, and now we have worked incredibly hard and we're bringing out what we call a Little Sun Diamond. We call it a diamond because we want people to say, "Okay, a diamond is valuable. So is energy. Energy is like a treasure."
The project started started in Ethiopia?
We started in Ethiopia. Also in Senegal. We're currently focusing on Senegal, Ethiopia, and South Africa. But we are, in total, in twelve countries in Africa. But you could say that Ethiopia is where we have the strongest network and infrastructure.
Where else have you been successful?
Every country in which we're working has its unique sort of economic circumstances and infrastructure.
Up until September last year, Zimbabwe was our most successful country because we had an incredible strong door-to-door salesman initiative in the outskirts of the capital of about a hundred and twenty young adults that had gone through a business education program working for us, selling the Little Sun in an organized, door-to-door salesman system. They organized their sales in collaboration with us, but we seed-funded them at the beginning, it grew, and it became a robust undertaking. But late last last year, the currency collapsed and the inflation went up, and suddenly these hundred and twenty self-employed Little Sun salespeople were unable to sustain it.
We're still in contact with them, and we're hoping that the economy is gonna stabilize. But the truth is that as much as the economy is actually growing in most of Sub-Saharan Africa, it is still a very unstable system due to poor governance and lack of strength in the private sector.
We're in the private sector because we believe very strongly in trade, but we're also working through the Little Sun Foundation, the small donation and philanthropic projects.
What is on the immediate horizon for the future of Little Sun?
We are working a lot with how to make energy even more affordable. What are the systems, what are the ways in which we can reach the people at the very bottom of the pyramid?
But that means we need to also be in the education of infrastructure. We need to work with the government, the ministers of education, and the local schools. So we have a very large focus on education and children. The transition to solar, or to sustainable energy, is going to come. So where will the oil go? The oil is gonna go to Africa. They're gonna try to lower the price, subsidize — so the competition with the oil industry will be fierce. If we want to actually introduce sustainable energy resources, and now Little Sun is a solar company, but it's also an energy company, and we are working in education, we are working in policy, we are trying to work on awareness. We can't quite say that we are campaigning, but in the spirit of the Little Sun there is a campaign built into it. We are really very active in vocalizing our mission.
Check out more videos from VICE:
What is the potential role of the individual -- someone in the VICE audience -- to do something toward the goal of Little Sun?
Thinking about the world won't change anything. What one needs to do is actually go out and say, "Where do I feel interconnected to the world?"
Little Sun won't succeed unless people empower us. We are, to a large extent, crowd-funded. We, I have thrown all my muscle into this. I've asked all of my friends to throw all their muscles into this, and taken my network to the extreme. So what I'm asking for the VICE viewers is the muscle. I'm not asking for the brain. We have too much brain already. The world has enough brains around. We need muscle. We need everybody to show they are not just thinking, they are actually doing.
Donate to the Little Sun Foundation to help deliver solar energy to the most vulnerable communities worldwide. If you choose to purchase a Little Sun Diamond for yourself, you also make the same product available to someone without access to electricity at a locally affordable price.


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Note "Mp3acquire pro"The writer ofMP3Doctorrecently renamed his "SuperMp3Normalizer" professionalgram to " Mp3gain professional ". i didn't cross the threshold this new professionalgram, please do not electronic mail me any help questions about it.should you're , here are the primary ceremonial variations between "Mp3acquire professional" and my, uh, "basic"(?) MP3acquire: "Mp3gain professional" does quantity normalizationinsidethe mp3, not simply between separate out mp3s. therefore in the event you really feel a music is too unresponsive in the beginning (or center, or finish), then it could possibly boost the quantity only for that half. fairly serene, if that's what you want.The changes "Mp3achieve professional" makes arenotundo-ready. as a way to make its superb-tuned advertjustments, it must re-fix the mp3 file.nonetheless, test it out if you're . however do not ask me any questions ;)


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EDSP 30243 - Academic Success in Special Education
Prerequisite: Admission to the Teacher Education Program or written permission of the dean, College of Education. Students enrolled in EC-4/Special Education Program must be concurrently enrolled in EDEC 30213 and EDEC 30223. This course focuses on procedures and processes for assessing and teaching basic academic skills including oral language, reading, written language and math skills to students with disabilities. Individual education program planning and monitoring also will be addressed.
Texas Christian University


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refuses to leave and do anything interesting, even at the urging of Dave Sim. Roleplaying Games In the Vorkosigan Saga RPG, it's recommended that the GM not include Miles Vorkosigan as an NPC. Kor agreed, citing original Chucks' "very flexible shank which would not be ideal for someone walking for eight to 10 hours per day and points to Converse's recently released Chuck Taylor II, which has a "more stable shank" and a remodeled interior and sole. Hes my favorite Doctor. Or just write for David! Get back to the plot, you bastards!
The film co-stars Dolph Lundgren, Burt Young, Talia Shire, Carl Weathers, Tony Burton, Brigitte Nielsen and Michael Pataki.
Rocky IV was the highest grossing sports movie for 24 years, before it was overtaken by The Blind Side.
It is the fourth and most financially successful entry in the.
He has apparently even "spoken" to some of them in real life. Even though the writer claims to be in control, the Doctor points out that the story is controlling him in a way clearly reminiscent of this trope. The narrator in Into the Woods has no more control over the events of the play than the characters, as proved when they band together and kill him. In the Doctor Who Expanded Universe book Who Killed Kennedy, a number of extremely unpleasant things happen to Dodo, a former companion of the Doctor. And practically guarantees that there will be no unilaterally happy endings. As a teenager, the budding actor learned that because there was already a David McDonald in the actors union, he needed to come up with an alternate moniker to pursue a professional acting career. While its hard to imagine that Tennant has ever had to deal with too many scathing reviews, it doesnt really matter to the actor: good or bad, he avoids reading them. I cannot stress this enough.". In her essay My Friend Mr Campion, Margery Allingham wrote that Albert Campion just appeared at the dinner table in The Crime at Black Dudley, and she had no idea who he was or what he was doing there, which was annoying as that was.
The story's control over him is even represented as mechanical parts and the writer lapsing from his usual genial personality into Robo Speak as he performs the story's bidding. "If you include him, he'll try to take over the plot from the PCs. Milholland of Something*Positive also plots out events years ahead of time or has notes of events he intends to do at certain points. Soap Opera Disease and dies, if the, happily Married couple break up for no apparent reason, or everything suddenly becomes very depressing, this is generally the writer's response to fan disbelief.


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HI i have point xyz and can make LAS dataset?
Correct Answer
I don't have any video explaining the steps, but if you can share the, let's say' first 100 records of the XYZ file, I can see if I can provide some more explanation on the steps to accomplish this.
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