Renova prices

Quick!. Everybody into the conference room. Today, we’re going to discuss what science has to say about some of the most memorable scenes from the enduring hit TV series, The Office.The Office ended in 2013, but the show continues to delight old fans and attract new ones on streaming services. The success of the Office Ladies podcast, hosted by Jenna Fischer (Pam) and Angela Kinsey (Angela), further affirms the show’s abiding popularity. It’s apparent that people won’t stop appreciating the endearing employees at the Dunder Mifflin Paper Company anytime soon.The outlandish scenes still make for interesting water cooler banter, and you might be wondering if there’s any truth to them.

Let’s take a coffee break and have an educational look at five classic moments from the show.Angela’s Beet Juice CleanseIn S6:E23, Dwight and Angela meet with a lawyer to discuss their childbearing contract. Item five, point “B” states that Angela must complete a “beet juice cleanse.” When Dwight asks for a stool sample to verify she is doing the cleanse, Angela flashes her red-stained teeth as proof instead.Juice cleansing is a controversial dietary trend. During the cleanse period, which is performed for 3–10 days to reportedly detox and lose weight, participants usually consume nothing but juices extracted from fruits and vegetables. Beets are a root vegetable and a good source of some nutrients such as folate, magnesium and vitamin C. Betalain pigments, which give beets the deep red color that stained Angela’s teeth, are antioxidants that also have anti-inflammatory effects.

Additionally, beets contain nitrates that widen blood vessels, which can reduce blood pressure and increase blood flow to the brain. One drawback to juicing is the loss of fiber, a key nutrient in this vegetable.Due to the sharp drop in caloric intake, people on a juice cleanse often lose a little weight. Unfortunately, it is typically gained back as soon as a normal diet resumes. Additionally, many juicers are likely to experience low blood sugar and depleted energy levels. Restricting the juice diet to a single fruit or vegetable will also deprive the individual of other vital nutrients, including protein.On occasion, especially in people with pre-existing conditions, juicing can lead to excess oxalate in the body, causing acute kidney stones or damage.

Given the deprivation of calories, the limited nutrients and the potential adverse effects, a juice cleanse would not be advisable during pregnancy or while trying to conceive.Incidentally, Dwight was not entirely off-base for requesting a stool sample to verify Angela’s compliance with the beet cleanse. In some people, the betalains can cause stools to darken and urine to redden (a side effect known as beeturia).Dwight’s “Hygiene Hypothesis”In S7:E7, Pam leads a discussion about how to minimize germs from being spread around the office. In response to hand sanitizers being set up in the workplace, Dwight protests, “The worst thing you can do for your immune system is to coddle it. €¦ If Sabre really cared about our well-being, they would set up hand desanitizing stations. A simple bowl at every juncture filled with dirt, vomit, fecal matter.”Dwight appears to be referring to the so-called “hygiene hypothesis,” which suggests that our modern germaphobic tendencies are detrimental to our immune system.

The idea is particularly applicable during childhood when the immune system is in its earliest stages of development. Failing to appropriately train the immune system during this critical period may cause it to malfunction. Without germs to fight, some think that the immune system might resort to attacking harmless things or the body, leading to allergies and autoimmune disorders, respectively.Supporting the idea are studies that have correlated the presence of microbes during childhood with decreased allergies. For example, some studies report a reduced incidence of hay fever in people who grew up on a farm as opposed to in a city. In some studies, this effect can be linked to animal exposure.

Even in a city environment, pets, particularly dogs, can have a protective effect from the development of allergies.It is doubtful that the hygiene hypothesis applies in adults, as the developmental window on the immune system has largely closed after 3 – 4 years of age. So, Dwight’s idea to dirty up the office is not only gross, but also scientifically unsound. Furthermore, the hygiene hypothesis is far from proven, and many confounding variables such as genes, diet and the prevalence of antibiotics and pollutants likely conspire to shape a person’s immune system.Since it was first proposed in 1989, the hygiene hypothesis has been controversial. Some scientists have argued that use of the word hygiene is an unfortunate misnomer that discourages people from being sanitary. Returning to an era of filth would only increase rates and detract from finding the real explanation behind the rise of asthma and allergy in developed societies.

A more recent version of the idea known as the “old friends” hypothesis distinguishes between good and bad microbes. It asserts that we should certainly protect ourselves and children from dangerous pathogens, such as those lurking in fecal matter, vomit or unclean food, but not be overly concerned about beneficial or harmless microbes that are routinely encountered. These are already present in and around our bodies and may be important for appropriately training the immune system.Rabies Awareness Fun RunIn S4:E1, Michael hits Meredith with his car, sending her to the hospital with a cracked pelvis. At the hospital, Meredith reveals she was also recently bitten by a bat, racoon and rat, on separate occasions. This prompts the doctors to begin treatment for rabies.

And it inspires Michael to organize the "Michael Scott's Dunder Mifflin Scranton Meredith Palmer Memorial Celebrity Rabies Awareness Pro-Am Fun Run Race for the Cure."But how big of a threat is rabies in reality?. Rabies is common enough in wildlife, but is rarely seen these days in domesticated animals and people living in developed nations. From 2009 to 2018, only 25 cases of human rabies were reported in the U.S.. That’s just one to three cases per year. Any mammal can be infected with rabies, but it is most frequently transmitted to humans by raccoons, skunks, bats and foxes.Rabies is a bullet-shaped renova that slowly creeps through the nerves until it finds the brain, where it causes a terrifying transformation that blurs the line between human and beast.

Rabid animals foam at the mouth and become ferociously aggressive. The sickness can turn a lamb into a lion. Also, as Michael Scott points out, people suffering from rabies develop an intense aversion to water known as hydrophobia.The rabies renova concentrates in saliva and can be transmitted through biting. You might think that a renova capable of such wizardry would be highly complex, but it contains only five genes. One of these genes makes a protein that appears to interfere with communication between cells in the brain, which likely contributes to the behavioral changes caused by rabies.

Fun fact. The hangover remedy known as “hair of the dog” has its origins in a supposed rabies treatment devised by the Roman naturalist Pliny the Elder. Pliny suggested rabies victims should “insert in the wound ashes of hairs from the tail of the dog that inflicted the bite.” Don’t try this. It does not work.As for Michael’s efforts, his Rabies Fun Run would have been more relevant prior to the 1880s, before Louis Pasteur developed the first rabies treatment. Or, in other parts of the world that face more cases of rabies.

Globally, rabies kills nearly 60,000 people each year, largely due to lack of resources and access to medical care.Lice Bug BombPediculus humanus capitis was the featured guest on S9:E10, causing an infestation across cubicles at 1725 Slough Avenue. While everyone assumed the head lice came from Meredith, the source was actually Pam, who contracted them from her daughter Cece.Lice are tiny insect parasites that take up residence on the scalp. These so-called skull vampires suck blood for nourishment and glue their eggs (nits) tightly to the hair. The insects can’t jump or fly, but can be passed between people who share hairbrushes, clips, bedding, towels, clothing or hats. The most common source of transmission is through direct contact with an infested person’s hair.

While head lice are an annoyance, they do not carry any known disease.Our friends on The Office put their heads together (not literally, thankfully) and offered several different solutions. Following Erin's advice, infected co-workers applied generous globs of mayonnaise to each other's hair to try to suffocate the lice. Meredith took a more radical approach and shaved her head. True to form, Dwight overreacts and attempts to rid the office of lice with a bug bomb grenade. Naturally, it explodes before he leaves the room, and the toxic fumes cause him to hallucinate and faint.Of all the solutions attempted, Meredith’s is most certain to work.

Depriving the lice of hair deprives them of a place to lay eggs, and the adults are easily washed away. But many people are not willing to sacrifice their locks. While it’s a popular home remedy, Erin’s idea to suffocate the lice with mayonnaise (petroleum jelly is also common) rarely works, according to (the aptly named) Mayo Clinic. And, as this episode illustrates, bug bombs are far more trouble than they are worth. Lice cannot survive without a host for more than a day, so there is no need to fumigate and risk exposure to dangerous chemicals.

More than 3,200 cases of bug bomb-related illnesses, including four human deaths, were reported in the U.S. Between 2007 and 2015.One effective way to treat lice is to use a shampoo containing an insecticide like permethrin. Permethrin is an insect neurotoxin that causes paralysis in the louse by disrupting sodium transport across its cellular membranes. Nit combs can be used in conjunction with the shampoo treatment to physically remove eggs unaffected by the insecticide. Multiple treatments are advised to ensure all of the lice have been eradicated.Kevin’s Stinky FeetJim and Pam’s wedding in S6:E4 was filled with unforgettable moments, including the revelation that Kevin has a serious foot odor issue.

Kevin left his shoes outside his hotel door to be cleaned, only to find that they had disappeared during the night. The hotel manager told him. €œMr. Malone, your shoes are gone. €¦ When the bag was opened by our shoeshine, the smell overcame him.

I, too, smelled them and made the choice that they must be thrown away. Incinerated, actually.”Scientists have sniffed out the cause of bromodosis (foot odor), and it can be traced to a bacterium called Brevibacterium linens. Our bodies are home to trillions of bacteria, likely more than 10,000 different species, that live on or inside us. B. Linens are harmless denizens of our skin, where they consume dead cells.

As they digest the dead skin cells, they release smelly sulfur-containing compounds called S-methyl thioesters as waste products.Sweaty feet create a moist and salty environment that allows this species of bacteria to thrive, generating pungent odors as they excrete more and more S-methyl thioesters. Incidentally, these are the same bacteria used to produce the rind of smelly cheeses like Limburger.Kevin could have reduced his foot odor by depriving the bacteria of the sweat they need to grow. He could have achieved this by wearing open-toed shoes whenever possible, using powder or carrying an extra pair of dry, fresh socks. There may also be additional hope on the horizon for folks like Kevin, cursed with industrial strength foot odor. Scientists recently found that socks coated in zinc oxide nanoparticles, which have potent antibacterial activity, are effective at preventing foot odor.Armed with that knowledge, you can now comfortably prop up your feet and marathon through all nine seasons of The Office.

Or, at least track down these standout episodes — with an eye toward science.For some people, the idea of going to the store without a mask right now is so shocking, they’re having stress dreams about it. But once the need to wear a face-covering every time we go shopping is over, our instinct to reach for our masks might not disappear entirely in the U.S. Living through the worst epidemic Americans have seen in a century might shift attitudes about long-term mask use, in part because what many people experienced during the renova is uniquely traumatic, says Isaac Fung, an epidemiologist at Georgia Southern University. €œIt’s probably a once-in-a-lifetime experience, even though there have been, and will be, skin carees that create an epidemic.” Who changes behaviors and how frequently they reach for their face coverings, however, could depend on a few factors.Lasting Effects of TraumaPart of why it’s possible that masks could become a more long-term fixture in the U.S. Is because elsewhere in the world, previous renovas had the same effect.

In 2003, the SARS outbreaks in parts of Asia, including China, Taiwan and South Korea, required mask-wearing. The shock of the SARS outbreaks and a cultural memory of what helped control them could partially explain why the transition to consistent mask use in some of these nations during skin care products was seamless compared to the U.S., Fung says. €œThey have both the fortunate and misfortune of the impact of SARS in 2003.” In between the renovas, consistent mask-wearing in parts of Asia evolved into an occasional polite choice someone might make if they had a cold or cough and were out in public. Masks, along with other skin care products protocols like hand washing and social distancing, can reduce the odds of someone spreading other illnesses like the seasonal flu. In the U.S., a similar scenario — a population scarred by a renova comes to realize how useful the masks are for other illnesses — might play out.

Granted, mask use has become political in the U.S. In a way it hasn’t in many other places, Fung points out. But throughout the renova, Pew Research Center surveys have shown that the partisan divide on masking behaviors lessened over time.Christos Lynteris, a social anthropologist at the University of St Andrews in Scotland, thinks future consistent mask use might stand a chance in part because the renova won’t end with one theatrical, celebratory announcement. If the health crisis was suddenly “over” one day, some people might reject masks completely from there on out. €œThrowing your mask away [could be] like you're unshackling yourself from the epidemic, which is over,” Lynteris says.

But it’s more likely the renova could see seasonal resurgences like the flu and draw out for a while. In that case, the longer battle with skin care could help individuals see masks as a more consistent part of life that comes with other health benefits. Too Close for ComfortThe realization of the perpetual benefits of masks might take hold in cities best, particularly if many residents rely on public transit, Fung says. When people don’t own cars and need to get around via packed buses or train cars, they spend a lot more time in close contact with other people. It’s hard to be in that environment daily and not see the value of a face covering, Fung says.

In large swathes of the U.S. Where people commute in their own cars or rely on relatively-empty public transportation, the appeal of wearing masks might not be as strong.Future mask use could also depend on how well people transform the face-covering into an effective but appealing — maybe even fashionable — accessory. If public health departments had wanted to make mask-wearing a more consistent part of long-term healthy behaviors, the institutions could have been more intentional about encouraging this transition, Lynteris says. €œYou need to allow people to adopt the mask as their own thing,” he says. Meeting with different communities and encouraging people to make masks look the way they want them to could make them more appealing.

In parts of Asia, people pulled off this exact transition with masks over the years. People sell and purchase masks that coordinate with outfits, and when it is part of the wardrobe, the face-covering becomes even more likely to be worn, Fung says. If covering faces in public persists for years to come, the well-meaning action would be more likely to be effective if people had a better idea of how to handle the masks. In fact, this is a part of mask education that Fung thinks could be improved right now during the renova. €œThis part of health education I do not see happening in the U.S.,” he says.

€œWe are only focusing on wearing it, not how to properly take it off or wash it.” Handling masks correctly can reduce the likelihood that any renova on the material doesn’t make its way into your nose or mouth. And while the CDC has guidelines on appropriate behavior, simply having online information available for those who search for it isn’t quite enough, Lynteris notes. Appropriate mask protocols are another topic public health officials should discuss with communities. A chance to ask questions — about when masks should be cleaned or how to dispose of them, for example — or even hear from others about the mistakes they’ve made can familiarize people with what they need to do themselves. €œIf you don’t use the mask correctly but think it's protecting you, you may be engaging in behavior where you put yourself and others at risk,” Lynteris says.

€œIt’s an important conversation we’re not having.”Alcohol consumption in the U.S. Surged in 2020. Booze delivery services gained popularity while market reports relayed information about higher sales volumes. Even academic surveys found people were drinking more — one sample representing roughly 1,500 American adults found that on average, three of every four individuals were pouring themselves a drink an extra day every month.Of course, having a beer one extra day of the month doesn’t necessarily mean someone is drinking too much. But when it comes to self-assessing booze habits, people tend to define the problem in a way that somehow leaves their own habits in good standing, says Patricia E.

Molina, the director of the Alcohol and Drug Abuse Center of Excellence at LSU Health Sciences Center New Orleans. €œWhat the lay public tends to do is pay attention or focus on one aspect that is convenient for their definition." Beyond Binge DrinkingTypically, people use the term binge drinking as a benchmark of whether or not they’ve had one too many. The term refers to booze consumption that brings someone’s blood alcohol content (BAC) to .08g/dl or above — the legal limit for driving in the U.S. Most men reach that value after having five drinks in two hours, while women typically reach it after having four drinks in the same time span. Binge drinking is the most common and deadly form of excessive drinking in the U.S., according to the CDC, as it’s associated with a wide range of health consequences.

Some stem from the impact alcohol has on the body, such as alcohol poisoning, while others are due to the way alcohol disrupts our ability to function, like injuries from car accidents. But even when people haven't reached excessive BAC levels, it doesn’t exempt them, or others, from harm. €œOne could make the argument that, okay, if I don't drink that much in two hours, but over a longer period of time, is that okay?. € Molina says. €œWell, not completely.” Besides binge drinking, the CDC also labels heavy drinking as a risky, harmful behavior, and is a concept Molina thinks should be a larger part of alcohol education campaigns.

Classified as eight or more drinks a week for women and 15 or more a week for men, heavy drinking is less likely to cause short-term issues, like car accidents. But over time, the habit can lead to a range of cancers, liver disease and heart problems, as well as depression and anxiety. So while someone might be able to drive their car after tailgating all Saturday, they may still have put away several drinks over the entire afternoon, Molina says, pushing the limit of what qualifies as a week of heavy drinking. Ultimately, the fewer drinks someone has, the better. To keep the health consequences of alcohol low, the official USDA dietary guidelines for 2020 to 2025 cap moderate drinking at two drinks a day for men and one drink a day for women.

However, the scientific advisory group that helps craft these guidelines has recommended that the cap be one drink a day for everyone. No Sense of StandardEven if someone was keeping track of their beer habit and trying to stick to these quantities, a lot of people don’t know or can’t estimate what qualifies as a single drink, Molina says. In the U.S., a standard drink contains 14 grams of alcohol. Since different kinds of drinks have a range of alcohol content, that serving size pans out to be 12 ounces of a 5 percent alcohol beer, five ounces of wine and one and a half ounces of liquor. These volumes don’t always match what someone might perceive as a single serving, like an oversized can of beer or a restaurant pour of wine, which is often closer to eight ounces, Molina says.

Studies have shown that people tend to overestimate what qualifies as a standard drink anyways, and when asked to pour out a single serving, are too generous. If people drink more than they think they do, then their threshold for what it takes to feel buzzed is likely higher than they thought, too. How people develop alcohol tolerance — where a given number of drinks has less of an effect on their ability to function over time — isn’t well understood by researchers, though there are likely genetic and social influences at work. But increasing tolerance is often associated with alcohol use disorder or alcohol dependence. The more someone drinks, Molina says, the more they need to achieve the relaxation or buzz they’re seeking through alcohol.

At the same time, “you increase the risk of falling into a pattern of drinking to avoid negative feelings,” she says.For anyone curious about the best ways to recalibrate their drinking patterns, Molina recommends Rethinking Drinking, a National Institute of Health resource that spells out serving sizes, how those compare to what standard drink containers hold, and what different drinking habits look like.Looking over every detail of the human body, male nipples may be the most obviously useless. In females, they are essential for delivering life-sustaining breast milk to newborns. In men, they serve, well, no apparent purpose. Virtually every other anatomical structure clearly furthers the enterprise of survival and reproduction, so why would we evolve one that doesn’t?. The answer may lie in a subtle misunderstanding of evolutionary theory’s fundamental assumption.

That living organisms change over time, becoming better adapted to their environments in response to the pressures of natural selection. Few scientific tenets are as certain as this one. But it’s easy to oversimplify that account and interpret it to mean that every modification in a species is adaptive, developed to fulfill a vital function.This tempting idea goes back as far as Erasmus Darwin, the grandfather of Charles Darwin, who considered adaptivity in his 1794 book Zoonomia. Or, the Laws of Organic Life, an early treatise on evolution. As far as he could see, all animal traits seem to have evolved toward greater utility, with one exception.

€œThe breasts and teats of all male quadrupeds, to which no use can now be assigned.” The Harmless NippleAs evolutionary theory progressed through another Darwin and beyond, biologists refined their understanding of its mechanisms. The evolutionary biologists Stephen Jay Gould and Richard Lewontin, in a highly influential (and provocative) 1979 paper, argued against the “Panglossian paradigm” of adaptation, which claims — like its indomitably optimistic namesake in Voltaire’s Candide — that evolution has crafted “the best of all possible worlds.”Gould and Lewontin contend that other factors besides natural selection control the operation of evolution, one being simply the lack of selective pressure against a trait. The male nipple doesn’t aid in survival and reproduction, but it doesn’t hinder, either. Why would natural selection do away with a harmless circle of flesh?. In another paper, in 1993, Gould addressed the nipple query specifically.

He wrote that of all the evolutionary conundrums for “well-informed nonscientific readers … no single item has evoked more puzzlement than the very issue that Erasmus Darwin chose as a primary challenge to his concept of pervasive utility — male nipples.” To Gould, the solution is straightforward. It requires “no adaptive explanation at all,” only the realization that evolution deals with more pressing matters than the superficial features of a man’s chest.Embryonic OriginsThere is, of course, a proximate explanation for the persistence of male nipples. In an odd way, Erasmus Darwin nearly glimpsed the truth in his own speculation two and a half centuries ago, when he suggested they were vestiges of an earlier stage of human evolution — a time “during the infancy of the world” when, he thought, all people were hermaphrodites. Males and females do, in fact, start from the same genetic blueprint. Embryos, in their first weeks, develop structures with the potential to become either male or female sex organs.

Only later do sex-specific hormones begin to mold those structures into either a clitoris or a penis, either functioning mammary glands or unproductive male nipples. “Males and females are not separate entities, shaped independently by natural selection,” Gould writes. €œBoth sexes are variants upon a single ground plan, elaborated in later embryology.” Nipples begin to appear before that sex differentiation begins, so males are stuck with them as they mature.It’s not entirely fair to say they serve no possible purpose, though. Under unusual conditions, like starvation or a spike in levels of the hormone prolactin, men can actually produce milk — to answer Robert DeNiro’s unforgettably funny and awkward inquiry in Meet the Parents. As Pulitzer-prize-winning author Jared Diamond wrote in a 1995 Discover article titled Father’s Milk, “Lactation, then, lies within a male mammal’s physiological reach.”Nipples are also highly sensitive, and research shows that in men as well as women they respond to sexual stimulation.

One study found that about half of men (and more than 80 percent of women) report nipple stimulation enhances their sexual arousal. Maybe these minor compensations, rather than a boost in physical and reproductive fitness, justify the male nipple’s existence.The latest results from the phase 3 skin care products treatments trials have been very positive. These have shown that vaccinating people with the gene for skin care spike protein can induce excellent protective immunity.The spike protein is the focus of most skin care products treatments as it is the part of the renova that enables it to enter our cells. renova replication only happens inside cells, so blocking entry prevents more renova being made. If a person has antibodies that can recognize the spike protein, this should stop the renova in its tracks.The three most advanced treatments (from Oxford/AstraZeneca, Pfizer/BioNTech and Moderna) all work by getting our own cells to make copies of the renova spike protein.

The Oxford treatment achieves this by introducing the spike protein gene via a harmless adenorenova vector. The other two treatments deliver the spike protein gene directly as mRNA wrapped in a nanoparticle. When our own cells make the spike protein, our immune response will recognize it as foreign and start making antibodies and T cells that specifically target it.However, the skin care renova is more complicated than just a spike protein. There are, in fact, four different proteins that form the overall structure of the renova particle. Spike, envelope (E), membrane (M) and nucleocapsid (N).

In a natural , our immune system recognizes all of these proteins to varying degrees. So how important are immune responses to these different proteins, and does it matter that the first treatments will not replicate these?. Parts of the skin care, including the N protein. (Credit. OSweetNature/Shutterstock)Following skin care , researchers have discovered that we actually make the most antibodies to the N protein – not the spike protein.

This is the same for many different renovaes that also have N proteins. But how N protein antibodies protect us from has been a long-standing mystery. This is because N protein is only found inside the renova particle, wrapped around the RNA. Therefore, N protein antibodies cannot block renova entry, will not be measured in neutralization assays that test for this in the lab, and so have largely been overlooked.New mechanism discoveredOur latest work from the MRC Laboratory of Molecular Biology in Cambridge has revealed a new mechanism for how N protein antibodies can protect against viral disease. We have studied another renova containing an N protein called lymphocytic choriomeningitis renova and shown a surprising role for an unusual antibody receptor called TRIM21.Whereas antibodies are typically thought to only work outside of cells, TRIM21 is only found inside cells.

We have shown that N protein antibodies that get inside cells are recognized by TRIM21, which then shreds the associated N protein. Tiny fragments of N protein are then displayed on the surface of infected cells. T cells recognize these fragments, identify cells as infected, then kill the cell and consequently any renova.We expect that this newly identified role for N protein antibodies in protecting against renova is important for skin care, and work is ongoing to explore this further. This suggests that treatments that induce N protein antibodies, as well as spike antibodies, could be valuable, as they would stimulate another way by which our immune response can eliminate skin care.Adding N protein to skin care treatments could also be useful because N protein is very similar between different skin carees – much more so than the spike protein. This means it’s possible that a protective immune response against skin care N protein could also offer some protection against other related skin carees, such as Mers.Another potential benefit that may arise from including N protein in skin care treatments is due to the low mutation rates seen in the N protein sequence.

Some changes to the sequence of skin care have been reported over the course of this renova, with the most significant changes occurring in the spike protein. There is some concern that if the spike sequence alters too much, then new treatments will be required. This could be similar to the current need for annual updating of influenza treatments. However, as the N protein sequence is much more stable than the spike, treatments that include a component targeting the N protein are likely to be effective for longer.The first wave of skin care treatments brings genuine hope that this renova can be controlled by vaccination. From here it will be an ongoing quest to develop even better treatments and ones that can remain effective in the face of an evolving renova.

Future treatments will probably focus on more than just the spike protein of skin care, and the N protein is a promising target to add to the current strategies being considered.This article is republished from The Conversation under a Creative Commons license. Read the original article..

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Increased economic integration and technological advancements in communication how to get renova and transportation over the past several decades have spurred growth in cross-national investment, migration and cultural exchange renova car. Nations, economies and people are increasingly interconnected and interdependent. Increasingly ‘globalised’ renova car. The concept of globalisation entered the mainstream vocabulary in the 1990s, but its history has been fraught with controversy.1 Primarily an economic process involving domestic deregulation, trade liberalisation and privatisation, globalisation can have profound social and cultural ramifications. Proponents highlight the economic benefits and renova car improved standards of living for many communities, while opponents of globalisation focus on the disproportionate channelling of wealth to larger Western nations and the further disempowerment of populations who lack the skills to meaningfully participate in this flow of information and resources.1Similarly, the globalisation of healthcare has also inspired competing interpretations and perspectives.

Historically, the globalisation of health has referred to the cross-border flow of healthcare professionals for employment, patients for medical services and public health and research measures across nations. These broad categories reflect the challenges renova car in defining this critical concept that informs social policy, drives change and impacts population health outcomes. More recently, the globalisation of medical education has been used to describe the transnational transfer of curricula, practices and accreditation standards, the global movements of faculty and medical trainees, and the establishment of international branches of medical schools and academic institutions.2 3 The importation of Western-based competencies and educational modalities has sparked discourse around the potential for ‘homogenisation and cultural dominance’ in medical education.2 4 Global accreditation requirements purport to establish standard outcomes and ensure minimum levels of competence, using standardised curricula and accreditation protocols.3 However, globalised medical education may not consistently align with local priorities and needs and has been criticised for imposing Western paradigms on non-dominant nations.2 For example, in India, Western influences predominate medical education, whereby curricula often focus on diseases not relevant to the community.5 In Southeast Asia, student-centred teaching approaches, including problem-based learning, were adopted even though they conflicted with longstanding cultural traditions and norms between students and teachers.6 As such, researchers and educators have expressed concerns that international medical education is overlooking important cultural nuances and is, instead, promoting standards that are Western, rather than truly global.2As medical educators in the Middle East, we have witnessed the effects of globalised medical education. Many students are sponsored by the government to train in medical schools and residency programmes in North America, Australia and Europe, with little consideration of the alignment renova car between the type and content of training received abroad and the needs of the home country to which they return. More recently, several Gulf countries have mandated the wide-scale implementation of US-based accreditation frameworks as part of graduate medical education reform efforts.3 7 8 This often translates to medical trainees that are taught by multinational faculty, using Western-based curricula and assessment methods, in fundamentally different sociocultural, economic and regulatory contexts.

The question renova car remains. How do educational systems maintain best practice and outcome standards while remaining responsive to the local needs?. Over the past decade, educational researchers worldwide have proposed glocalisation as a potential answer.Glocalisation, a neologism combining the terms globalisation and localisation, describes the adaptation of international standards to local needs and cultures.4 By glocalising curricula, accreditation standards and renova car educational practices, trainees learn to provide global standards of care that address local health priorities. The ultimate goal of the glocalisation of medical education is the advancement of population health outcomes and system responsiveness to local health needs. Glocalisation efforts in the medical education literature highlight three main themes renova car.

(1) local adaptation of accreditation standards, (2) exploration of educational methodologies towards glocalisation and (3) identification of challenges facing glocalisation efforts. We will review each of these areas in an attempt to further describe renova car this construct.Much of the globalisation in medical education literature deals with the adoption of accreditation standards. Many countries in Europe, Asia and the Middle East have adopted the competency-based framework of the Royal College of Canada.9 When the US-based Accreditation Council for Graduate Medical Education offered international accreditation services in 2010, several countries rapidly adopted its model and standards.3 Not surprisingly, glocalisation efforts have focused on ensuring local relevance of related standards and processes. Research has shown that these efforts are diverse and often require renova car input from multiple stakeholders. For example, Ho et al describe four categories of deviation between global accreditation standards and medical schools attempting to glocalise their local accreditation systems in Taiwan, Japan and South Korea.4 These include structural differences of medical education in the national context (such as programme length, entry requirements and school governance), differences http://donhughesdevelopment.com/?page_id=8 requiring adaptation of standards to conform to local regulatory environments, developmental trajectory differences representing the influence of contextual events on medical education and aspirational differences reflecting local priorities and focuses.4Other attempts to glocalise have focused on educational competencies, rather than accreditation standards.

Several authors renova car have questioned the applicability of Western definitions of medical professionalism to their local contexts. In this regards, glocalisation efforts towards the development of culturally relevant medical professionalism curricula represent a common area of study.10 For example, in the United Arab Emirates, we implemented a novel methodology towards glocalising medical professionalism, employing several consensus-gathering techniques. The resultant definition identified additional domains to Western definitions of professionalism that incorporated culturally relevant constructs, including spirituality in professional practice and the role of family and community in patient care decisions.10 Many other educational constructs, such as leadership, communication skills and medical ethics, cannot renova car be directly imported from one country to another but require local adaptation.Finally, when considering the process of glocalisation, studies reveal that educational leaders must give due consideration to the complexity of challenges encountered. These include diverse or conflicting views on educational objectives and scopes, a lack of representation of the diverse perspectives of the local context, a lack of a shared mental model of competence, misalignment of educational requirements and health system factors and the influence of power relationships and decision-makers on the glocalisation process.4 Ensuring diverse representation in glocalisation efforts is critical to fostering consensus, mitigating the challenges identified, facilitating the consideration of contextual factors and leveraging local networks of support.All education is local. However, for the foreseeable future, healthcare and health education will be renova car impacted by an increasingly interconnected world.

This serves to highlight the critical importance of ensuring that medical education institutions remain accountable to the communities they serve. These seemingly discordant responsibilities renova car are reconciled through deliberate glocalisation efforts. If the ultimate goal of medical education is the production of a competent healthcare workforce, equipped with universal practice standards that can meet local population health needs, glocalisation practices must be viewed as essential components of educational standards, and should be adopted by medical educators, accreditation and regulatory bodies and healthcare institutions in the global arena.Ethics statementsPatient consent for publicationNot required.AbstractThe concept of continuity in medical education reflects the progressive professional and personal development that physicians need in education. The aim of this study renova car is investigating the views of the residents about the adequacy of undergraduate and postgraduate education in the context of preparing them for the next stage and their perceptions about the transition period. This phenomenological study was conducted at Hacettepe University Medical School.

The study group consisted of medical and renova car surgical sciences residents in the first year and last year of postgraduate medical education. Four focus group interviews were held with the participation of 21 residents. The participants emphasised that practising with real renova car patients under supervision by taking an active role in healthcare teams was important for their preparation for the next stage in their carrier. However, their educational experiences during undergraduate medical education differed in community-based education, scientific research training, learning in small groups, internship and guidance of clinical educators. The transition period has been expressed with the concepts of identity change, high responsibilities and expectations required by the new identity, renova car adaptation to the healthcare team, institution, and health system, meeting the expectations in an overly busy work environment, and feelings of incompetence.

Participants pointed out that curriculum, which was declared and taught, educational environments, assessment approaches, consultancy systems and practices differed between the clinical departments. In line with the principles of competency-based education, practices related to the development and assessment of the competencies with all professional aspects in postgraduate medical education can renova car be strengthened.Medical education &. Training.

Increased economic integration and http://www.jamiegianna.com/courses-2/ technological advancements in communication and transportation over the past several decades have renova prices spurred growth in cross-national investment, migration and cultural exchange. Nations, economies and people are increasingly interconnected and interdependent. Increasingly ‘globalised’ renova prices. The concept of globalisation entered the mainstream vocabulary in the 1990s, but its history has been fraught with controversy.1 Primarily an economic process involving domestic deregulation, trade liberalisation and privatisation, globalisation can have profound social and cultural ramifications.

Proponents highlight the economic benefits and improved standards of living for many renova prices communities, while opponents of globalisation focus on the disproportionate channelling of wealth to larger Western nations and the further disempowerment of populations who lack the skills to meaningfully participate in this flow of information and resources.1Similarly, the globalisation of healthcare has also inspired competing interpretations and perspectives. Historically, the globalisation of health has referred to the cross-border flow of healthcare professionals for employment, patients for medical services and public health and research measures across nations. These broad categories reflect the challenges in defining this critical concept that informs social renova prices policy, drives change and impacts population health outcomes. More recently, the globalisation of medical education has been used to describe the transnational transfer of curricula, practices and accreditation standards, the global movements of faculty and medical trainees, and the establishment of international branches of medical schools and academic institutions.2 3 The importation of Western-based competencies and educational modalities has sparked discourse around the potential for ‘homogenisation and cultural dominance’ in medical education.2 4 Global accreditation requirements purport to establish standard outcomes and ensure minimum levels of competence, using standardised curricula and accreditation protocols.3 However, globalised medical education may not consistently align with local priorities and needs and has been criticised for imposing Western paradigms on non-dominant nations.2 For example, in India, Western influences predominate medical education, whereby curricula often focus on diseases not relevant to the community.5 In Southeast Asia, student-centred teaching approaches, including problem-based learning, were adopted even though they conflicted with longstanding cultural traditions and norms between students and teachers.6 As such, researchers and educators have expressed concerns that international medical education is overlooking important cultural nuances and is, instead, promoting standards that are Western, rather than truly global.2As medical educators in the Middle East, we have witnessed the effects of globalised medical education.

Many students are sponsored renova prices by the government to train in medical schools and residency programmes in North America, Australia and Europe, with little consideration of the alignment between the type and content of training received abroad and the needs of the home country to which they return. More recently, several Gulf countries have mandated the wide-scale implementation of US-based accreditation frameworks as part of graduate medical education reform efforts.3 7 8 This often translates to medical trainees that are taught by multinational faculty, using Western-based curricula and assessment methods, in fundamentally different sociocultural, economic and regulatory contexts. The question renova prices remains. How do educational systems maintain best practice and outcome standards while remaining responsive to the local needs?.

Over the past decade, educational researchers worldwide have proposed glocalisation as a potential answer.Glocalisation, a neologism combining the terms globalisation and localisation, describes the adaptation of international standards to local needs and cultures.4 By glocalising curricula, accreditation standards and educational practices, trainees learn to provide global standards of renova prices care that address local health priorities. The ultimate goal of the glocalisation of medical education is the advancement of population health outcomes and system responsiveness to local health needs. Glocalisation efforts in renova prices the medical education literature highlight three main themes. (1) local adaptation of accreditation standards, (2) exploration of educational methodologies towards glocalisation and (3) identification of challenges facing glocalisation efforts.

We will review each of these areas in an attempt to further renova prices describe this construct.Much of the globalisation in medical education literature deals with the adoption of accreditation standards. Many countries in Europe, Asia and the Middle East have adopted the competency-based framework of the Royal College of Canada.9 When the US-based Accreditation Council for Graduate Medical Education offered international accreditation services in 2010, several countries rapidly adopted its model and standards.3 Not surprisingly, glocalisation efforts have focused on ensuring local relevance of related standards and processes. Research has shown that these efforts are diverse and often renova prices require input from multiple stakeholders. For example, Ho et al describe four categories of deviation between global accreditation standards and medical schools attempting to glocalise their local accreditation systems in Taiwan, Japan and South Korea.4 These include structural differences of medical education in the national context (such as programme length, entry requirements and school governance), differences requiring adaptation of standards to conform to local regulatory environments, developmental trajectory differences representing the influence of contextual events on medical education and aspirational differences reflecting local priorities and focuses.4Other attempts to glocalise have focused on educational competencies, rather than accreditation standards.

Several authors renova prices have questioned the applicability of Western definitions of medical professionalism to their local contexts. In this regards, glocalisation efforts towards the development of culturally relevant medical professionalism curricula represent a common area of study.10 For example, in the United Arab Emirates, we implemented a novel methodology towards glocalising medical professionalism, employing several consensus-gathering techniques. The resultant definition identified additional domains to Western definitions renova prices of professionalism that incorporated culturally relevant constructs, including spirituality in professional practice and the role of family and community in patient care decisions.10 Many other educational constructs, such as leadership, communication skills and medical ethics, cannot be directly imported from one country to another but require local adaptation.Finally, when considering the process of glocalisation, studies reveal that educational leaders must give due consideration to the complexity of challenges encountered. These include diverse or conflicting views on educational objectives and scopes, a lack of representation of the diverse perspectives of the local context, a lack of a shared mental model of competence, misalignment of educational requirements and health system factors and the influence of power relationships and decision-makers on the glocalisation process.4 Ensuring diverse representation in glocalisation efforts is critical to fostering consensus, mitigating the challenges identified, facilitating the consideration of contextual factors and leveraging local networks of support.All education is local.

However, for the foreseeable future, healthcare and health education will be impacted by an renova prices increasingly interconnected world. This serves to highlight the critical importance of ensuring that medical education institutions remain accountable to the communities they serve. These seemingly discordant responsibilities are reconciled through deliberate glocalisation renova prices efforts. If the ultimate goal of medical education is the production of a competent healthcare workforce, equipped with universal practice standards that can meet local population health needs, glocalisation practices must be viewed as essential components of educational standards, and should be adopted by medical educators, accreditation and regulatory bodies and healthcare institutions in the global arena.Ethics statementsPatient consent for publicationNot required.AbstractThe concept of continuity in medical education reflects the progressive professional and personal development that physicians need in education.

The aim of this study is investigating the views of renova prices the residents about the adequacy of undergraduate and postgraduate education in the context of preparing them for the next stage and their perceptions about the transition period. This phenomenological study was conducted at Hacettepe University Medical School. The study group consisted of medical and surgical sciences residents in the first year and last year of postgraduate renova prices medical education. Four focus group interviews were held with the participation of 21 residents.

The participants emphasised that practising with real patients under supervision by taking an active role in healthcare renova prices teams was important for their preparation for the next stage in their carrier. However, their educational experiences during undergraduate medical education differed in community-based education, scientific research training, learning in small groups, internship and guidance of clinical educators. The transition period has been expressed with the concepts of identity change, high responsibilities and expectations required by the new identity, adaptation to the healthcare team, institution, and health system, meeting the expectations in an overly busy renova prices work environment, and feelings of incompetence. Participants pointed out that curriculum, which was declared and taught, educational environments, assessment approaches, consultancy systems and practices differed between the clinical departments.

In line with the principles of competency-based education, practices related to the development and assessment of the competencies with all professional aspects in postgraduate medical education can be strengthened.Medical renova prices education &. Training.

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Renova homes

July 15, renova homes 2021Contact. Office of CommunicationsPhone. 202-693-1999OSHA, National Demolition Association renova homes enter allianceto protect safety, health of demolition contractors WASHINGTON, DC – The U.S. Department of Labor’s Occupational Safety and Health Administration and the National Demolition Association recently signed a two-year alliance agreement to protect the safety and health of workers in demolition and related industries. The goal of the alliance is to renova homes improve demolition industry safety by providing training and targeting industry-specific hazards.

The alliance will focus on developing best practices in power plant demolition and providing agency staff with training on best practices related to deconstructing and dismantling building components for reuse, repurposing, recycling and waste management. €œPlanning for a demolition job is as important as doing the work,” said Acting Assistant Secretary of Labor renova homes for Occupational Safety and Health Jim Frederick. €œWe look forward to working with the National Demolition Association to help reinforce the importance of making adequate preparations for bringing down a building, training all workers on industry hazards and safety precautions in a language they understand, providing appropriate personal protective equipment and complying with OSHA standards.” Demolition work involves many of the hazards associated with construction, but includes additional hazards from unknown factors, such as changes or modifications that alter the original design, materials hidden within structural components, and unknown strengths/weaknesses of construction materials, as well as hazards created by the demolition methods used. The National Demolition Association is a non-profit trade association comprising nearly 400 member companies nationally and internationally. The association provides educational resources on structural renova homes demolition and dismantlement, industrial recovery, recycling, architectural salvage decontamination, asbestos abatement and nuclear clean-up.

Learn more about demolition hazards. # # renova homes # U.S. Department of Labor news materials are accessible at http://www.dol.gov. The department’s Reasonable renova homes Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay).July 15, 2021Contact.

Office of CommunicationsPhone renova homes. 202-693-1999OSHA schedules meeting of the Advisory Committee onConstruction Safety and Health for August 11 WASHINGTON, DC – The U.S. Department of Labor’s Occupational Safety and Health Administration has scheduled a meeting of the Advisory Committee on Construction Safety and Health for 1 p.m. To 5 p.m renova homes. EDT, Wednesday, Aug.

11, 2021 renova homes. The meeting will include agency updates and remarks from Acting Assistant Secretary of Labor for Occupational Safety and Health Jim Frederick, OSHA Directorate of Construction industry updates, a discussion of the OSHA Construction Focus Four Hazards, an ACCSH Workgroup discussion, and a public comment period. Comments and renova homes requests to speak must be submitted electronically at http://www.regulations.gov, the Federal eRulemaking Portal, by Tuesday, July 27. Include Docket Number OSHA-2021-0004 on all submissions. Attendance at this meeting renova homes will be virtual only.

Telecommunication information will be posted in the docket and on the ACCSH webpage. Read the Federal Register notice for submission details. ACCSH advises renova homes the Secretary of Labor and Assistant Secretary of Labor for Occupational Safety and Health on construction standards and policy matters. It was established under the Contract Work Hours and Safety Standards Act and the Occupational Safety and Health Act of 1970. Learn more about renova homes ACCSH.

# # # U.S. Department of renova homes Labor news materials are accessible at http://www.dol.gov. The department’s Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay)..

July 15, hop over to here 2021Contact renova prices. Office of CommunicationsPhone. 202-693-1999OSHA, National Demolition Association enter allianceto protect safety, health of demolition contractors WASHINGTON, DC – renova prices The U.S. Department of Labor’s Occupational Safety and Health Administration and the National Demolition Association recently signed a two-year alliance agreement to protect the safety and health of workers in demolition and related industries. The goal of the alliance is to improve demolition industry safety by providing training and renova prices targeting industry-specific hazards.

The alliance will focus on developing best practices in power plant demolition and providing agency staff with training on best practices related to deconstructing and dismantling building components for reuse, repurposing, recycling and waste management. €œPlanning for a demolition job is as important as doing the work,” said Acting Assistant Secretary of Labor renova prices for Occupational Safety and Health Jim Frederick. €œWe look forward to working with the National Demolition Association to help reinforce the importance of making adequate preparations for bringing down a building, training all workers on industry hazards and safety precautions in a language they understand, providing appropriate personal protective equipment and complying with OSHA standards.” Demolition work involves many of the hazards associated with construction, but includes additional hazards from unknown factors, such as changes or modifications that alter the original design, materials hidden within structural components, and unknown strengths/weaknesses of construction materials, as well as hazards created by the demolition methods used. The National Demolition Association is a non-profit trade association comprising nearly 400 member companies nationally and internationally. The association provides educational resources renova prices on structural demolition and dismantlement, industrial recovery, recycling, architectural salvage decontamination, asbestos abatement and nuclear clean-up.

Learn more about demolition hazards. # # # renova prices U.S. Department of Labor news materials are accessible at http://www.dol.gov. The department’s renova prices Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay).July 15, 2021Contact.

Office of renova prices CommunicationsPhone. 202-693-1999OSHA schedules meeting of the Advisory Committee onConstruction Safety and Health for August 11 WASHINGTON, DC – The U.S. Department of Labor’s Occupational Safety and Health Administration has scheduled a meeting of the Advisory Committee on Construction Safety and Health for 1 p.m. To 5 p.m renova prices. EDT, Wednesday, Aug.

11, 2021 renova prices. The meeting will include agency updates and remarks from Acting Assistant Secretary of Labor for Occupational Safety and Health Jim Frederick, OSHA Directorate of Construction industry updates, a discussion of the OSHA Construction Focus Four Hazards, an ACCSH Workgroup discussion, and a public comment period. Comments and requests to speak must be submitted electronically at http://www.regulations.gov, the Federal eRulemaking Portal, by Tuesday, July 27 renova prices. Include Docket Number OSHA-2021-0004 on all submissions. Attendance at this renova prices meeting will be virtual only.

Telecommunication information will be posted in the docket and on the ACCSH webpage. Read the Federal Register notice for submission details. ACCSH advises the Secretary of Labor and Assistant Secretary of Labor for Occupational renova prices Safety and Health on construction standards and policy matters. It was established under the Contract Work Hours and Safety Standards Act and the Occupational Safety and Health Act of 1970. Learn more renova prices about ACCSH.

# # # U.S. Department of renova prices Labor news materials are accessible at http://www.dol.gov. The department’s Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay)..

Renova zero arctic blue

AbstractGene fusion, a genomic event that generates a novel gene from two independent genes, has long been known to be implicated in tumourigenesis and renova zero arctic blue cancer progression. It has thus served renova zero arctic blue as a diagnostic and prognostic biomarker in cancer, as well as an ideal therapeutic target in cancer therapy. Gene fusion can arise from chromosomal rearrangement and alternative splicing of transcripts, resulting in deregulation of proto-oncogenes or creation of an oncogenic novel gene. Largely facilitated by next generation sequencing technologies, a plethora of novel gene fusions have been identified in a variety of cancers, which leaves us the challenge of functionally characterising renova zero arctic blue these candidate gene fusions. In this review, we summarise the molecular mechanisms, the oncogenic consequences and the therapeutic implications of verified gene fusions.

We also discuss recent studies on gene fusions in both common and rare subtypes of ovarian tumours and how these findings can be renova zero arctic blue translated to cancer therapies to benefit patients carrying these gene fusions.medical oncologygeneticsIntroductionIdentification of a germline pathogenic TP53 (MIM. *191170) variant in a patient with cancer has drastic medical impacts.1 Indeed, in TP53 variant carriers, chemotherapy and radiotherapy have been shown to contribute to the development of subsequent primary cancers, the incidence of which is remarkably high (above 40%).1–4 Therefore, in these patients, surgical treatment should be prioritised and radiotherapy and chemotherapy avoided, if possible, or at least carefully discussed in terms of benefit:risk ratio between risk of recurrence and risk of inducing second primary tumours. Furthermore, TP53 variant renova zero arctic blue carriers should have specific surveillance protocols, including annual whole-body MRI,5 6 whose efficiency for early tumour detection has recently been shown by numerous studies.5–14Interpretation of germline TP53 variants, which are mainly missense variants, remains particularly complex. Whereas germline variants of TP53 were initially detected in Li-Fraumeni syndrome (LFS, MIM#151623),15–17 our perception of cancers related to germline alterations of TP53 has drastically evolved over time.1 2 18 19 The presence of a disease-causing germline variant should be considered in patients fulfilling Chompret criteria, which were sequentially updated and extended.1 The question of germline TP53 variant interpretation is becoming a growing concern in the field because the renova zero arctic blue TP53 gene is currently included in many cancer gene panels, and the number of TP53 tests performed in patients not fulfilling the criteria mentioned earlier has increased exponentially. 20 21Classification of TP53 variants, in agreement with the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines, is based on several items, including frequency of the variant in the general population (gnomAD.

Https://gnomad.broadinstitute.org/), segregation data, bioinformatics predictions and functional assays developed in yeast or human cancer cell lines.22 One of the first assays commonly used for TP53 missense variant interpretation was developed in yeast and is based on the expression of TP53 cDNA in strains containing reporter plasmids with different p53 binding sites.23 In this renova zero arctic blue assay, p53 variants are classified as functional, not functional or partially functional if the transcriptional activity is conserved for some but not all yeast reporter plasmids (http://p53.iarc.fr/). More recently, two teams have developed in human cancer cell lines high throughput p53 functional assays.24 25 Kotler et al24 generated a synthetic library of TP53 variants located within the p53 DNA-binding domain and quantified the antiproliferative activity of these variants in the p53-null H1299 cancer cell line. In this assay, TP53 variants are categorised as ‘wild-type TP53-like renova zero arctic blue variant’ (functional) or ‘disrupting’ (non-functional). In another assay, Giacomelli et al25 generated by saturation mutagenesis a TP53 library and tested the ability of the variants (1) to restore the survival of the p53-null A459 cell line exposed to high doses of DNA damaging agents, in order to detect loss of function (LOF) variants and (2) to induce in p53-wild-type A459 cells resistance to Nutlin-3, in order to detect variants with dominant negative effect (DNE).We previously developed, in Epstein-Barr renova-immortalised lymphocytes, a p53 functional assay exploring the transcriptional activity of the protein underlying its tumour suppressor activity.26 This assay is based on the exposure of cells to DNA damaging agents followed by the measurement of the p53 transcriptional response.27 28 With this assay, we showed that pathogenic TP53 variant carriers exhibit a constitutive defect in the transcriptional response to DNA damage, establishing a biological endophenotype associated with germline pathogenic variants.27 28 Compared with the other assays, its main advantage is to evaluate the impact of heterozygous variants in the genetic context of the patients. Its main disadvantage is that it requires EBV immortalisation, which is time-consuming and, therefore, not suited for a rapid classification and interpretation of TP53 variants in medical practice.Therefore, despite the different tools indicated previously and before the completion in the future of curated international databases, interpretation of germline renova zero arctic blue TP53 variants remains challenging in clinical practice.

This prompted us to develop a p53 functional assay derived from the previous one but performed on fresh blood samples and suitable for rapid interpretation and medical management of patients. We show here that this assay can accurately detect pathogenic variants and can be used to reallocate unclassified variants by integrating the results to the classification strategy.22 Furthermore, this assay revealed that a TP53 polymorphism (rs78378222), present in 1.7% of the European population, compromises p53 functional activity with the same magnitude as a heterozygous null variant, when carried on renova zero arctic blue both alleles.MethodsCell culture and treatmentEBV-immortalised cell lines were maintained in RPMI 1640 medium (GIBCO. Life Technologies, Carlsbad, California, USA) with 10% fetal calf renova zero arctic blue serum (Invitrogen, Life Technologies) and 1% L-glutamine (Invitrogen) at 37°C with 5% CO2. Cells were seeded in duplicate in 12-well plates (Corning, New York, USA) at a density of 106 cells/well. Cells were treated or not with 200 ng/mL renova zero arctic blue (0.3 µM final concentration) of doxorubicin (Sigma Aldrich, St.

Louis, Missouri, USA) for 8 hours. Cells were washed with 1× PBS and harvested for RNA extraction.Peripheral blood renova zero arctic blue mononuclear cell (PBMC) isolation and cultureBlood samples were collected in EDTA tubes and kept for 2 days at room temperature before PBMC isolation on a lymphocyte separation medium (Eurobio, Evry, France). From 2.5 to 10.0 mL of blood per patient was used for PBMC isolation. Cell number and cell viability were assessed on a NanoEnTek Adam automatic cell counter renova zero arctic blue with the AccuChip Kit (ScienceTEC, Villebon-sur-Yvette, France). One million cells were seeded per well in a 24-well plate and were let to grow for 48 hours in a lymphocyte activating medium (Chromosome Medium P, AmpliTech, Compiègne, France).

At least two wells were seeded per patient (treated and untreated) and duplicates or renova zero arctic blue triplicates were performed whenever possible. Cells were treated with 800 ng/mL of doxorubicin for 8 hours, washed with 1× PBS, harvested and RNA extraction was performed using the NucleoSpin RNA XS kit (Macherey Nagel, Düren, Germany) renova zero arctic blue according to the manufacturer’s instructions and quantified using a UV-VIS ND-1000 spectrophotometer (Biocompare, Nanodrop Technologies, USA).RNA-SeqFour control EBV cell lines wild-type for TP53 and four heterozygous TP53-mutant cell lines, corresponding to three canonical dominant negative missense variants (p.(Arg175His), p.(Arg248Trp) and p.(Arg273His)) and one complete deletion of the TP53 locus, were treated or not with doxorubicin. RNA was extracted using the Nucleospin RNAII kit (Macherey Nagel). Libraries were prepared using the NEBNext Ua Directional RNA Library Kit for Illumina (NEB, Ipswich, USA) and NGS sequencing of the libraries was performed on an Illumina NextSeq500 (Illumina, San Diego, USA) using 2*75 bp sequencing to generate 50M read pairs renova zero arctic blue on average per sample. Experiments were performed in triplicates.

Bioinformatic analysis was carried out using an in-house automated pipeline AURIGA that uses the STAR V.2.5.3a tool for alignment, FeatureCounts tool V.1.5.2 for read counting and DESeq2 V.1.18.1 for statistical analysis.Selection of biomarkers indicative of p53-transcriptional activityNew biomarkers were selected among the transcripts strongly up-egulated by doxorubicin in control cells but not in the cells harbouring heterozygous TP53 renova zero arctic blue alterations. CEP170B (NM_015005), PODXL (MIM*602632, NM_001018111), RRAD (MIM*179503, NM_004165), GLS2 (MIM*606365, NM_013267), CABYR (MIM*612135, NM_012189), TP53I3 (MIM*605171, NM_004881), EPS8L2 (MIM*614988, NM_022772), SULF2 (MIM*610013, NM_001161841), SESN1 (MIM*606103, NM_014454) and FHL2 (MIM*602633, NM_201555). Three control transcripts with a renova zero arctic blue steady expression across all conditions and genotypes and expressed at the same level as the selected targets were also selected. TBP (MIM*600075, NM_003194), RIC8B (MIM*609147, NM_001330145) and MPP5 (MIM*606958, NM_022474.3). An internal control of treatment efficacy was included renova zero arctic blue.

PLK1 (MIM*602098, NM_005030.5), whose transcript is downregulated by doxorubicin treatment renova zero arctic blue both in wild-type and mutant cells.Reverse transcription–quantitative multiplex PCR of short fluorescent fragment (RT-QMPSF)Reverse transcription (RT) was performed on 100 ng of total RNA using the Verso cDNA Synthesis Kit (Thermo Scientific, Waltham, USA). RT-QMPSF was performed on 1.5 µL of RT using Diamond Taq DNA polymerase (Kaneka Eurogentec, Seraing, Belgium), 6% Dymethyl sulfoxide and 26 PCR cycles (94°C. 30 s/58°C. 1 min/72°C. 30 s).

Primer sequences are listed in online supplemental table 1. Amplicons were analysed on an ABI Prism 3500 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) using GeneScan 3.7 software.Supplemental materialReverse transcription–multiplex ligation probe amplification (RT-MLPA)RT-MLPA probes were pooled at a concentration of 1 fmol/µL each in 10 mM Tris/1 mM EDTA. Probe sequences are given in online supplemental table 1. RT (6.5 µL), probe mixture (1.5 µL) and SALSA-MLPA buffer (1.5 µL, MRC-Holland, Amsterdam, The Netherlands) were mixed before denaturation (95°C, 2 min) and hybridisation (60°C, 1 hour). Ligation was performed at 54°C for 15 min, adding 32 µL of ligation mixture, and heated 5 min at 98°C.

Then, 2.5 µL of the ligation was added to 7.5 µL of a Q5Hot Start High-Fidelity 2X Master Mix (NEB) supplemented with universal fluorescent PCR primers. PCR was performed using 35 cycles (94°C. 30 s/58°C. 30 s/72°C. 30 s).

Amplicons were analysed on an ABI Prism 3500 Genetic Analyzer using GeneScan V.3.7 software.Calculation of p53 functionality score and p53 mRNA ratioThe RT-MLPA or RT-QMPSF profiles of doxorubicin-treated and untreated cells were superimposed after adjusting the control amplicons to the same height. In the treated condition, the peak height of each of the 10 p53 target genes was measured and divided by the sum of the heights of the three control genes. This value was then divided by the same ratio calculated in the untreated condition. In the assay, the mean of the 10 values defines the p53 functionality score. The final p53 functionality score is the mean of the scores obtained in RT-MLPA and RT-QMPSF assays.

The p53 mRNA levels were expressed as a ratio of the normal values obtained for 3 TP53 wild-type control individuals. The efficacy of the genotoxic treatment was assessed by calculating a PLK1 (MIM*602098) ratio (treated/untreated) normalised with the three controls, which should be less than 0.5.ResultsDevelopment of a rapid p53 functional assay performed on bloodThe rationale of the assay is that p53 acts as a powerful transcriptional inductor when DNA damage occurs and that the common deleterious impact of pathogenic variants is the alteration of this transcriptional activity.26 To develop a functional assay directly performed on patient’s fresh blood, we first optimised the quantitative assay that we had previously developed in EBV-immortalised cell lines.27 28 To this aim, we performed a new comparative transcriptomic analysis using RNA-Seq, including non-polyadenylated RNAs. Four control EBV cell lines wild type for TP53 and four patients with LFS EBV cell lines were compared in the context of genotoxic stress induced by doxorubicin treatment. We selected 10 biomarkers corresponding to p53 targets involved in different biological pathways controlled by p53, such as cell adhesion and migration, cellular response to stress, apoptosis, cytoskeleton organisation, glycolysis or regulation of other metabolic pathways. To normalise the results, we selected three transcripts with a steady expression across all conditions and genotypes.

All these biomarkers were then included in two quantitative assays based on RT-MLPA and RT-QMPSF. To detect in the same assay the potential effect of variants on the TP53 transcript levels, we added different amplicons or probes corresponding to TP53 cDNA. As a defect in treatment efficacy would result in a low functionality score leading to the misinterpretation of a wild-type genotype as a mutant one, we also integrated in the assays an internal control of treatment efficacy. After exposure to doxorubicin, cells were harvested and the RT-MLPA and RT-QMPSF assays were performed in parallel for each sample to increase the robustness of the assay. An arbitrary functionality score was calculated from the induction score of the 10 p53 targets.

The p53 RNA levels were evaluated and expressed as a percentage of the mean levels obtained for three wild-type TP53 individuals. This new quantitative assay, based on both RT-QMPSF and RT-MLPA, was first validated on 31 lymphoblastoid cell lines derived from patients with LFS harbouring different germline heterozygous TP53 variants (online supplemental table 2).Supplemental materialWe then set up the conditions allowing the assay to be performed directly on the patients’ peripheral blood. Blood was collected in conventional EDTA tubes and kept at room temperature for 2 days to mimic sample shipping delays. PBMCs were isolated and cultured for 48 hours in a lymphocyte activating medium. Under these conditions, a strong p53 transcriptional response could be monitored in wild-type individuals (figure 1), indicating that testing p53 function directly on patients’ blood cells was feasible.P53 functional assay on peripheral blood.

(A) Schematic representation of the blood p53 functional assay workflow. (B,C) Typical RT-QMPSF (B) and RT-MLPA (C) results obtained for individual 15 with a wild-type TP53 genotype. The fluorescent profiles of doxorubicin-treated cells (red line) and untreated cells (blue line) were superimposed using the three control amplicons (RIC8B, TBP and MPP5). The horizontal bars indicate for each p53 target gene the level of expression in untreated cells. Treatment efficacy was evaluated by the transcriptional repression of the PLK1 marker (Plk1 treated/untreated ratio below 0.5).

In the treated condition, the peak height of each of the 10 p53 target genes was measured and divided by the sum of the heights of the three control genes. This value was divided by the same ratio calculated in the untreated condition to yield an arbitrary p53 functionality score. The p53 mRNA levels were expressed as a ratio of the normal values obtained for three control individuals. PBMC, peripheral blood mononuclear cell. RT-MLPA, reverse transcription–multiplex ligation probe amplification.

RT-QMPSF, reverse transcription–quantitative multiplex PCR of short fluorescent fragment." data-icon-position data-hide-link-title="0">Figure 1 P53 functional assay on peripheral blood. (A) Schematic representation of the blood p53 functional assay workflow. (B,C) Typical RT-QMPSF (B) and RT-MLPA (C) results obtained for individual 15 with a wild-type TP53 genotype. The fluorescent profiles of doxorubicin-treated cells (red line) and untreated cells (blue line) were superimposed using the three control amplicons (RIC8B, TBP and MPP5). The horizontal bars indicate for each p53 target gene the level of expression in untreated cells.

Treatment efficacy was evaluated by the transcriptional repression of the PLK1 marker (Plk1 treated/untreated ratio below 0.5). In the treated condition, the peak height of each of the 10 p53 target genes was measured and divided by the sum of the heights of the three control genes. This value was divided by the same ratio calculated in the untreated condition to yield an arbitrary p53 functionality score. The p53 mRNA levels were expressed as a ratio of the normal values obtained for three control individuals. PBMC, peripheral blood mononuclear cell.

RT-MLPA, reverse transcription–multiplex ligation probe amplification. RT-QMPSF, reverse transcription–quantitative multiplex PCR of short fluorescent fragment.p53 functional analysis of patient’s blood cells with different TP53 genotypesWe then applied the p53 functional assay on blood samples sent to our laboratory for TP53 molecular analysis (NGS screening of the 11 exons complemented by QMPSF). Molecular and functional analyses were performed in parallel, in double blind conditions. We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.

This sample reflects the real-life recruitment of our diagnostic laboratory as it includes unaffected individuals as well as individuals affected by cancer who may have undergone different chemotherapy treatments. Molecular analyses revealed that 51 individuals had no detectable germline TP53 variant. For these 51 individuals, the mean p53 functionality score measured was 12.7 (13.6 for the RT-QMPSF assay and 11.9 for the RT-MLPA assay) with a range of 7.5–22.8 (online supplemental table 3 and figure 2). The mean observed p53 mRNA levels were 93% with a range of 74%–125% (online supplemental table 3). In eight tested individuals, molecular analysis revealed seven distinct TP53 variants which could be considered as likely pathogenic or pathogenic based on their ClinVar classification or their truncating nature (table 1).

All the variants tested were confirmed to be germline heterozygous variants. For these eight patients, the assay yielded a reduced score compared with the wild-type individuals (mean 4.8, range 3.1–7.1. Table 1 and figure 2). In the patients with missense variants, p53 mRNA levels were above 75%. In contrast, p53 mRNA was clearly reduced in patients harbouring frameshift or splice variants (mean 58%, table 1 and figure 2) probably reflecting the activity of the nonsense-mediated mRNA decay.Supplemental materialView this table:Table 1 Interpretation of germline TP53 variants integrating the blood p53 functional assayp53 functional scores and mRNA level ratios in individuals with wild-type TP53 or with germline TP53 variants.

(A) p53 functionality scores obtained in 51 wild-type TP53 individuals, compared with the scores obtained for nine samples from eight individuals carrying a classified TP53 variant (online supplemental table 3) using a Mann-Whitney non-parametric test. (B) Comparison of the p53 mRNA ratios obtained in 51 wild-type TP53 individuals and in samples carrying a missense (five samples) or a truncating variant of TP53 (four samples), using a Kruskal-Wallis test with Dunns post-test (p=0.0031). ***PFigure 3 Impact of the heterozygous and homozygous TP53 c.*1175A>C variation on p53 pre-mRNA 3′ end processing. (A) Schematic representation of the TP53 3′ end region. The c.*1175A>C variant is predicted to yield at least two different transcripts.

The upper one corresponds to the normal transcript with pre-mRNA cleavage and polyadenylation, and the lower one to longer transcript that extends after the poly-A signal. €˜Exon 11’ primers amplify both transcripts, while ‘postpoly-A’ primers specifically amplify the longer transcripts. As postpoly-A primers could also amplify gDNA, primers ‘exon 7’ and ‘exon 10’, which are specific to gDNA, were added to the reaction in order to monitor DNA contamination. (B) RT-QMPSF result obtained for the index case’s father (individual 58, S1. Table 1 and online supplemental table 3) carrying the variant TP53 c.723del, p.(Cys242Alafs*5).

The profile (in red) was superimposed on the profile of a control individual wild type for TP53 (in blue), using the control amplicons RIC8B and TBP. (C) RT-QMPSF result obtained for the index case’s mother (individual 76, S1. Table 1 and online supplemental table 3) carrying the c.*1175A>C variant at the homozygous state. (D) RT-QMPSF result for the index case (individual 77, online supplemental table 3) carrying the c.723del, p.(Cys242Alafs*5) variant and the c.*1175A>C in trans. Red arrows indicate the appearance of longer p53 transcripts.

The horizontal bars show the reduction of the normal p53 transcript level, as compared with the control. RT-QMPSF, reverse transcription–quantitative multiplex PCR of short fluorescent fragment.DiscussionThe interpretation of germline TP53 variants in patients with cancer is critical and should be performed before starting treatment considering their medical impact. The main objective of our assay was to provide a fast functional classification of rare uncharacterised variants in order to help clinicians with decision-making. Compared with the previous assay that we developed in EBV-immortalised lymphocytes,27 28 this blood assay does not require long-term cell culture and the results can be obtained within 1 week, fulfilling the timing required for diagnostic practice. The only constraint is to perform it within 48 hours after blood sampling in order to obtain robust results.

Under these conditions, we were able to successfully analyse samples sent from other European countries.Our assay fulfils most of the recommendations recently published by the Clinical Genome Resource Sequence Variant Interpretation working group regarding the clinical validity of functional assays29. (1) compared with the previously described p53 functional assays that test in vitro either cloned cDNA in yeast or artificial mutant libraries in cancer cell lines,23–25 this blood assay is performed in clinical samples in the patients’ genetic context. (2) the assay evaluates the transcriptional activity of p53 and not a specific domain of the protein. (3) it analyses simultaneously the impact of the variant on protein function and mRNA levels. (4) it was validated using 51 wild-type TP53 controls and 8 patients with seven distinct pathogenic or likely-pathogenic TP53 variants.

And finally, (5) results show the robustness of the assay. Indeed, as shown in table 1, for 12 tested variants, we were able to perform the assay on EBV-immortalised cell lines and the results were very similar. Moreover, for five individuals, two different blood samples were tested and yielded similar results (table 1), and two variants (c.844C>T, p.(Arg282Trp). C.847C>T, p.(Arg283Cys)) were tested on two different individuals’ blood with concordant results (4.8 vs 5.0 and 5.3 vs 6.4).We observed among the wild-type TP53 individuals a wide range of functionality scores (7.5–22.8). This probably suggests that there is a variability of the p53-mediated transcriptional response to DNA damage in the general population, although no obvious impact of age, clinical status or sex could be observed.

The thresholds used in this study could be refined by testing additional deleterious variants. Despite this variability, all pathogenic/likely pathogenic variants generated low p53 functionality scores, and variants resulting in premature stop codons were also detected by a clear reduction of p53 mRNA levels. In addition, our assay allows testing of non-missense variants such as in frame indels. It should be highlighted that none of the previously published functional assays can be considered as a gold-standard method to classify germline TP53 variants.23–25 Therefore, no available p53 functional assay can be used to calibrate the blood assay. Indeed, as illustrated in table 1, discordant results were obtained for variants unambiguously classified in ClinVar as pathogenic or likely pathogenic.

In particular, the founder Brazilian p.(Arg337His), an example of a variant with low penetrance, highlights the limits of the available tools. Whereas segregation data performed on large Brazilian pedigrees have clearly shown that this variant is pathogenic,34 bioinformatic predictions and functional analyses35 are conflicting (table 1). Our blood functional assay clearly shows that this variant alters the transcriptional activity of p53, although to a lesser extent than DNE missense variations, highlighting the limits of functional assays based on overexpression of cDNA. This result was confirmed in four additional patients carrying this variant using EBV cell lines (table 1).The blood functional assay performed on PBMC harbouring unclassified variants led us to consider 12 variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del)) as ‘functionally abnormal’, some with high impact. The interpretation is particularly challenging for p.(Pro72His), p.(Arg110His), p.(Arg158Cys), p.(Arg283Cys) and p.(Asp352Tyr) variants, as they were considered in yeast assays as functional or partially functional, and the Giacomelli assay classified them as not LOF_not DNE or was not conclusive.

The low functionality score observed for p.(Arg110His) was confirmed in an EBV cell line derived from the patient and confirmed in two EBV cell lines from other patients carrying this variant. The result for the p.(Asp352Tyr) variant was confirmed on a second blood sample and with an EBV cell line derived from another patient also carrying this variant. The effect of p.(Arg283Cys) was also confirmed in EBV cell lines derived from the patient and from three additional patients with the same variant (table 1).The clinical utility of the p53 functional assay is highlighted by the p.(Pro191Arg) variant. This variant was initially detected in a child with medulloblastoma at 2 years of age and whose brother died from a fibrosarcoma. Presymptomatic testing revealed that an unaffected brother (18 months), the mother and two maternal aunts were also carriers.

We were then requested to evaluate this variant, and the functional assay performed in the maternal aunt (individual 65, online supplemental table 3) clearly showed that this variant does not alter the p53 transcriptional activity (table 1 and online supplemental table 3). Considering this result, segregation analysis was performed on the brother’s fibrosarcoma sample, revealing the absence of the variant and consolidating the conclusion of a non-pathogenic variant.Our results show that this blood functional assay is also able to detect TP53 variations outside the coding regions, which are the only regions commonly analysed. Thanks to this assay, we discovered that the unaffected mother of an index case was homozygous for the polymorphic c.*1175A>C variant, and we show that this variant decreases p53 mRNA by altering the polyadenylation signal and produces longer transcripts extending beyond the poly-A site, as previously reported.30 When present on both alleles, this variant impacts p53 functionality with the same magnitude as a germline pathogenic TP53 variant. This prompted us to recommend breast MRI every year for this unaffected adult relative. We had the opportunity to perform the assay on EBV-immortalised lymphocytes harbouring only this heterozygous variant, and we observed a normal score (data not shown), suggesting that the heterozygous c.*1175A>C variant alone is insufficient to alter p53 function.

The comparison of the p53 functional scores observed in the index case who developed a high-grade glioma at 5 years of age and harbours the null c.723del, p.(Cys242Alafs*5) variant and in trans the polymorphic c.*1175A>C variant, and in her father carrying only the TP53 null variant suggests that the c.*1175A>C variant may act as a genetic modifier in pathogenic TP53 variant carriers and could increase the risk of glioma in carriers, as previously shown in the general population.30–33In summary, we suggest that our blood p53 functional assay should be a useful tool not only for the rapid interpretation of germline TP53 variants of unknown significance in clinical practice, in complement to the previously developed assays, but also for the indirect detection of cryptic alterations within regulatory regions impacting p53 function.Data availability statementAll data relevant to the study are included in the article or uploaded as supplementary information. Deidentified participant data are available from thierry.frebourg@chu-rouen.fr.Ethics statementsPatient consent for publicationNot required.AcknowledgmentsThe authors are grateful to their French and European colleagues for providing clinical information and sending blood samples for TP53 analysis. The authors are indebted to Philippe Ruminy (Inserm U1245, Comprehensive Cancer Centre Becquerel, Rouen) for advices on the reverse transcription–multiplex ligation probe amplification experiments and to Nikki Sabourin-Gibbs (Rouen University Hospital) for her assistance in editing the manuscript..

AbstractGene fusion, a genomic event that generates a novel gene from two independent genes, has long renova prices been known to be implicated in tumourigenesis and cancer progression. It has thus renova prices served as a diagnostic and prognostic biomarker in cancer, as well as an ideal therapeutic target in cancer therapy. Gene fusion can arise from chromosomal rearrangement and alternative splicing of transcripts, resulting in deregulation of proto-oncogenes or creation of an oncogenic novel gene. Largely facilitated by next generation sequencing technologies, renova prices a plethora of novel gene fusions have been identified in a variety of cancers, which leaves us the challenge of functionally characterising these candidate gene fusions. In this review, we summarise the molecular mechanisms, the oncogenic consequences and the therapeutic implications of verified gene fusions.

We also discuss recent studies on gene fusions in both common and rare subtypes of ovarian tumours and how these findings can be translated to cancer therapies to benefit patients renova prices carrying these gene fusions.medical oncologygeneticsIntroductionIdentification of a germline pathogenic TP53 (MIM. *191170) variant in a patient with cancer has drastic medical impacts.1 Indeed, in TP53 variant carriers, chemotherapy and radiotherapy have been shown to contribute to the development of subsequent primary cancers, the incidence of which is remarkably high (above 40%).1–4 Therefore, in these patients, surgical treatment should be prioritised and radiotherapy and chemotherapy avoided, if possible, or at least carefully discussed in terms of benefit:risk ratio between risk of recurrence and risk of inducing second primary tumours. Furthermore, TP53 variant carriers should have specific surveillance protocols, including annual whole-body MRI,5 6 whose efficiency for early tumour detection has recently renova prices been shown by numerous studies.5–14Interpretation of germline TP53 variants, which are mainly missense variants, remains particularly complex. Whereas germline variants of TP53 were initially detected in Li-Fraumeni syndrome (LFS, MIM#151623),15–17 our perception of cancers related to germline alterations of TP53 has drastically evolved over time.1 2 18 19 The presence of a disease-causing germline variant should be considered in patients fulfilling Chompret criteria, which were sequentially updated and extended.1 The question of germline TP53 variant interpretation is becoming a growing concern in the field because the TP53 gene renova prices is currently included in many cancer gene panels, and the number of TP53 tests performed in patients not fulfilling the criteria mentioned earlier has increased exponentially. 20 21Classification of TP53 variants, in agreement with the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines, is based on several items, including frequency of the variant in the general population (gnomAD.

Https://gnomad.broadinstitute.org/), segregation data, bioinformatics predictions and functional assays developed in yeast or human cancer cell lines.22 One of the first assays commonly used for TP53 missense variant interpretation was developed in yeast and is based on the expression of TP53 cDNA in strains containing reporter plasmids with different p53 binding sites.23 In this assay, p53 variants are classified as functional, renova prices not functional or partially functional if the transcriptional activity is conserved for some but not all yeast reporter plasmids (http://p53.iarc.fr/). More recently, two teams have developed in human cancer cell lines high throughput p53 functional assays.24 25 Kotler et al24 generated a synthetic library of TP53 variants located within the p53 DNA-binding domain and quantified the antiproliferative activity of these variants in the p53-null H1299 cancer cell line. In this renova prices assay, TP53 variants are categorised as ‘wild-type TP53-like variant’ (functional) or ‘disrupting’ (non-functional). In another assay, Giacomelli et al25 generated by saturation mutagenesis a TP53 library and tested the ability of the variants (1) to restore the survival of the p53-null A459 cell line exposed to high doses of DNA damaging agents, in order to detect loss of function (LOF) variants and (2) to induce in p53-wild-type A459 cells resistance to Nutlin-3, in order to detect variants with dominant negative effect (DNE).We previously developed, in Epstein-Barr renova-immortalised lymphocytes, a p53 functional assay exploring the transcriptional activity of the protein underlying its tumour suppressor activity.26 This assay is based on the exposure of cells to DNA damaging agents followed by the measurement of the p53 transcriptional response.27 28 With this assay, we showed that pathogenic TP53 variant carriers exhibit a constitutive defect in the transcriptional response to DNA damage, establishing a biological endophenotype associated with germline pathogenic variants.27 28 Compared with the other assays, its main advantage is to evaluate the impact of heterozygous variants in the genetic context of the patients. Its main disadvantage is that it requires EBV immortalisation, which is time-consuming and, therefore, not suited for a rapid classification and interpretation of TP53 variants in medical practice.Therefore, despite the different tools indicated previously and renova prices before the completion in the future of curated international databases, interpretation of germline TP53 variants remains challenging in clinical practice.

This prompted us to develop a p53 functional assay derived from the previous one but performed on fresh blood samples and suitable for rapid interpretation and medical management of patients. We show here that this assay renova prices can accurately detect pathogenic variants and can be used to reallocate unclassified variants by integrating the results to the classification strategy.22 Furthermore, this assay revealed that a TP53 polymorphism (rs78378222), present in 1.7% of the European population, compromises p53 functional activity with the same magnitude as a heterozygous null variant, when carried on both alleles.MethodsCell culture and treatmentEBV-immortalised cell lines were maintained in RPMI 1640 medium (GIBCO. Life Technologies, Carlsbad, California, USA) with 10% fetal calf serum (Invitrogen, Life Technologies) and 1% L-glutamine (Invitrogen) at 37°C with renova prices 5% CO2. Cells were seeded in duplicate in 12-well plates (Corning, New York, USA) at a density of 106 cells/well. Cells were treated or not with 200 ng/mL (0.3 µM final concentration) of doxorubicin (Sigma Aldrich, St renova prices.

Louis, Missouri, USA) for 8 hours. Cells were washed with 1× PBS and harvested renova prices for RNA extraction.Peripheral blood mononuclear cell (PBMC) isolation and cultureBlood samples were collected in EDTA tubes and kept for 2 days at room temperature before PBMC isolation on a lymphocyte separation medium (Eurobio, Evry, France). From 2.5 to 10.0 mL of blood per patient was used for PBMC isolation. Cell number and cell viability were assessed on a NanoEnTek Adam automatic cell counter with the AccuChip Kit (ScienceTEC, renova prices Villebon-sur-Yvette, France). One million cells were seeded per well in a 24-well plate and were let to grow for 48 hours in a lymphocyte activating medium (Chromosome Medium P, AmpliTech, Compiègne, France).

At least renova prices two wells were seeded per patient (treated and untreated) and duplicates or triplicates were performed whenever possible. Cells were treated with 800 ng/mL of doxorubicin renova prices for 8 hours, washed with 1× PBS, harvested and RNA extraction was performed using the NucleoSpin RNA XS kit (Macherey Nagel, Düren, Germany) according to the manufacturer’s instructions and quantified using a UV-VIS ND-1000 spectrophotometer (Biocompare, Nanodrop Technologies, USA).RNA-SeqFour control EBV cell lines wild-type for TP53 and four heterozygous TP53-mutant cell lines, corresponding to three canonical dominant negative missense variants (p.(Arg175His), p.(Arg248Trp) and p.(Arg273His)) and one complete deletion of the TP53 locus, were treated or not with doxorubicin. RNA was extracted using the Nucleospin RNAII kit (Macherey Nagel). Libraries were prepared using the NEBNext Ua Directional RNA Library Kit for Illumina (NEB, Ipswich, USA) and NGS sequencing of the libraries was performed on an renova prices Illumina NextSeq500 (Illumina, San Diego, USA) using 2*75 bp sequencing to generate 50M read pairs on average per sample. Experiments were performed in triplicates.

Bioinformatic analysis was carried out using an in-house automated pipeline AURIGA that uses the STAR V.2.5.3a tool for alignment, FeatureCounts tool V.1.5.2 for read counting and DESeq2 V.1.18.1 for statistical analysis.Selection of biomarkers indicative of p53-transcriptional activityNew biomarkers were selected among the transcripts strongly up-egulated by doxorubicin in control cells but not in the renova prices cells harbouring heterozygous TP53 alterations. CEP170B (NM_015005), PODXL (MIM*602632, NM_001018111), RRAD (MIM*179503, NM_004165), GLS2 (MIM*606365, NM_013267), CABYR (MIM*612135, NM_012189), TP53I3 (MIM*605171, NM_004881), EPS8L2 (MIM*614988, NM_022772), SULF2 (MIM*610013, NM_001161841), SESN1 (MIM*606103, NM_014454) and FHL2 (MIM*602633, NM_201555). Three control transcripts with a steady expression across all conditions and genotypes renova prices and expressed at the same level as the selected targets were also selected. TBP (MIM*600075, NM_003194), RIC8B (MIM*609147, NM_001330145) and MPP5 (MIM*606958, NM_022474.3). An internal renova prices control of treatment efficacy was included.

PLK1 (MIM*602098, NM_005030.5), whose transcript is downregulated by doxorubicin treatment both in wild-type and mutant renova prices cells.Reverse transcription–quantitative multiplex PCR of short fluorescent fragment (RT-QMPSF)Reverse transcription (RT) was performed on 100 ng of total RNA using the Verso cDNA Synthesis Kit (Thermo Scientific, Waltham, USA). RT-QMPSF was performed on 1.5 µL of RT using Diamond Taq DNA polymerase (Kaneka Eurogentec, Seraing, Belgium), 6% Dymethyl sulfoxide and 26 PCR cycles (94°C. 30 s/58°C. 1 min/72°C. 30 s).

Primer sequences are listed in online supplemental table 1. Amplicons were analysed on an ABI Prism 3500 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) using GeneScan 3.7 software.Supplemental materialReverse transcription–multiplex ligation probe amplification (RT-MLPA)RT-MLPA probes were pooled at a concentration of 1 fmol/µL each in 10 mM Tris/1 mM EDTA. Probe sequences are given in online supplemental table 1. RT (6.5 µL), probe mixture (1.5 µL) and SALSA-MLPA buffer (1.5 µL, MRC-Holland, Amsterdam, The Netherlands) were mixed before denaturation (95°C, 2 min) and hybridisation (60°C, 1 hour). Ligation was performed at 54°C for 15 min, adding 32 µL of ligation mixture, and heated 5 min at 98°C.

Then, 2.5 µL of the ligation was added to 7.5 µL of a Q5Hot Start High-Fidelity 2X Master Mix (NEB) supplemented with universal fluorescent PCR primers. PCR was performed using 35 cycles (94°C. 30 s/58°C. 30 s/72°C. 30 s).

Amplicons were analysed on an ABI Prism 3500 Genetic Analyzer using GeneScan V.3.7 software.Calculation of p53 functionality score and p53 mRNA ratioThe RT-MLPA or RT-QMPSF profiles of doxorubicin-treated and untreated cells were superimposed after adjusting the control amplicons to the same height. In the treated condition, the peak height of each of the 10 p53 target genes was measured and divided by the sum of the heights of the three control genes. This value was then divided by the same ratio calculated in the untreated condition. In the assay, the mean of the 10 values defines the p53 functionality score. The final p53 functionality score is the mean of the scores obtained in RT-MLPA and RT-QMPSF assays.

The p53 mRNA levels were expressed as a ratio of the normal values obtained for 3 TP53 wild-type control individuals. The efficacy of the genotoxic treatment was assessed by calculating a PLK1 (MIM*602098) ratio (treated/untreated) normalised with the three controls, which should be less than 0.5.ResultsDevelopment of a rapid p53 functional assay performed on bloodThe rationale of the assay is that p53 acts as a powerful transcriptional inductor when DNA damage occurs and that the common deleterious impact of pathogenic variants is the alteration of this transcriptional activity.26 To develop a functional assay directly performed on patient’s fresh blood, we first optimised the quantitative assay that we had previously developed in EBV-immortalised cell lines.27 28 To this aim, we performed a new comparative transcriptomic analysis using RNA-Seq, including non-polyadenylated RNAs. Four control EBV cell lines wild type for TP53 and four patients with LFS EBV cell lines were compared in the context of genotoxic stress induced by doxorubicin treatment. We selected 10 biomarkers corresponding to p53 targets involved in different biological pathways controlled by p53, such as cell adhesion and migration, cellular response to stress, apoptosis, cytoskeleton organisation, glycolysis or regulation of other metabolic pathways. To normalise the results, we selected three transcripts with a steady expression across all conditions and genotypes.

All these biomarkers were then included in two quantitative assays based on RT-MLPA and RT-QMPSF. To detect in the same assay the potential effect of variants on the TP53 transcript levels, we added different amplicons or probes corresponding to TP53 cDNA. As a defect in treatment efficacy would result in a low functionality score leading to the misinterpretation of a wild-type genotype as a mutant one, we also integrated in the assays an internal control of treatment efficacy. After exposure to doxorubicin, cells were harvested and the RT-MLPA and RT-QMPSF assays were performed in parallel for each sample to increase the robustness of the assay. An arbitrary functionality score was calculated from the induction score of the 10 p53 targets.

The p53 RNA levels were evaluated and expressed as a percentage of the mean levels obtained for three wild-type TP53 individuals. This new quantitative assay, based on both RT-QMPSF and RT-MLPA, was first validated on 31 lymphoblastoid cell lines derived from patients with LFS harbouring different germline heterozygous TP53 variants (online supplemental table 2).Supplemental materialWe then set up the conditions allowing the assay to be performed directly on the patients’ peripheral blood. Blood was collected in conventional EDTA tubes and kept at room temperature for 2 days to mimic sample shipping delays. PBMCs were isolated and cultured for 48 hours in a lymphocyte activating medium. Under these conditions, a strong p53 transcriptional response could be monitored in wild-type individuals (figure 1), indicating that testing p53 function directly on patients’ blood cells was feasible.P53 functional assay on peripheral blood.

(A) Schematic representation of the blood p53 functional assay workflow. (B,C) Typical RT-QMPSF (B) and RT-MLPA (C) results obtained for individual 15 with a wild-type TP53 genotype. The fluorescent profiles of doxorubicin-treated cells (red line) and untreated cells (blue line) were superimposed using the three control amplicons (RIC8B, TBP and MPP5). The horizontal bars indicate for each p53 target gene the level of expression in untreated cells. Treatment efficacy was evaluated by the transcriptional repression of the PLK1 marker (Plk1 treated/untreated ratio below 0.5).

In the treated condition, the peak height of each of the 10 p53 target genes was measured and divided by the sum of the heights of the three control genes. This value was divided by the same ratio calculated in the untreated condition to yield an arbitrary p53 functionality score. The p53 mRNA levels were expressed as a ratio of the normal values obtained for three control individuals. PBMC, peripheral blood mononuclear cell. RT-MLPA, reverse transcription–multiplex ligation probe amplification.

RT-QMPSF, reverse transcription–quantitative multiplex PCR of short fluorescent fragment." data-icon-position data-hide-link-title="0">Figure 1 P53 functional assay on peripheral blood. (A) Schematic representation of the blood p53 functional assay workflow. (B,C) Typical RT-QMPSF (B) and RT-MLPA (C) results obtained for individual 15 with a wild-type TP53 genotype. The fluorescent profiles of doxorubicin-treated cells (red line) and untreated cells (blue line) were superimposed using the three control amplicons (RIC8B, TBP and MPP5). The horizontal bars indicate for each p53 target gene the level of expression in untreated cells.

Treatment efficacy was evaluated by the transcriptional repression of the PLK1 marker (Plk1 treated/untreated ratio below 0.5). In the treated condition, the peak height of each of the 10 p53 target genes was measured and divided by the sum of the heights of the three control genes. This value was divided by the same ratio calculated in the untreated condition to yield an arbitrary p53 functionality score. The p53 mRNA levels were expressed as a ratio of the normal values obtained for three control individuals. PBMC, peripheral blood mononuclear cell.

RT-MLPA, reverse transcription–multiplex ligation probe amplification. RT-QMPSF, reverse transcription–quantitative multiplex PCR of short fluorescent fragment.p53 functional analysis of patient’s blood cells with different TP53 genotypesWe then applied the p53 functional assay on blood samples sent to our laboratory for TP53 molecular analysis (NGS screening of the 11 exons complemented by QMPSF). Molecular and functional analyses were performed in parallel, in double blind conditions. We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.

This sample reflects the real-life recruitment of our diagnostic laboratory as it includes unaffected individuals as well as individuals affected by cancer who may have undergone different chemotherapy treatments. Molecular analyses revealed that 51 individuals had no detectable germline TP53 variant. For these 51 individuals, the mean p53 functionality score measured was 12.7 (13.6 for the RT-QMPSF assay and 11.9 for the RT-MLPA assay) with a range of 7.5–22.8 (online supplemental table 3 and figure 2). The mean observed p53 mRNA levels were 93% with a range of 74%–125% (online supplemental table 3). In eight tested individuals, molecular analysis revealed seven distinct TP53 variants which could be considered as likely pathogenic or pathogenic based on their ClinVar classification or their truncating nature (table 1).

All the variants tested were confirmed to be germline heterozygous variants. For these eight patients, the assay yielded a reduced score compared with the wild-type individuals (mean 4.8, range 3.1–7.1. Table 1 and figure 2). In the patients with missense variants, p53 mRNA levels were above 75%. In contrast, p53 mRNA was clearly reduced in patients harbouring frameshift or splice variants (mean 58%, table 1 and figure 2) probably reflecting the activity of the nonsense-mediated mRNA decay.Supplemental materialView this table:Table 1 Interpretation of germline TP53 variants integrating the blood p53 functional assayp53 functional scores and mRNA level ratios in individuals with wild-type TP53 or with germline TP53 variants.

(A) p53 functionality scores obtained in 51 wild-type TP53 individuals, compared with the scores obtained for nine samples from eight individuals carrying a classified TP53 variant (online supplemental table 3) using a Mann-Whitney non-parametric test. (B) Comparison of the p53 mRNA ratios obtained in 51 wild-type TP53 individuals and in samples carrying a missense (five samples) or a truncating variant of TP53 (four samples), using a Kruskal-Wallis test with Dunns post-test (p=0.0031). ***PFigure 3 Impact of the heterozygous and homozygous TP53 c.*1175A>C variation on p53 pre-mRNA 3′ end processing. (A) Schematic representation of the TP53 3′ end region. The c.*1175A>C variant is predicted to yield at least two different transcripts.

The upper one corresponds to the normal transcript with pre-mRNA cleavage and polyadenylation, and the lower one to longer transcript that extends after the poly-A signal. €˜Exon 11’ primers amplify both transcripts, while ‘postpoly-A’ primers specifically amplify the longer transcripts. As postpoly-A primers could also amplify gDNA, primers ‘exon 7’ and ‘exon 10’, which are specific to gDNA, were added to the reaction in order to monitor DNA contamination. (B) RT-QMPSF result obtained for the index case’s father (individual 58, S1. Table 1 and online supplemental table 3) carrying the variant TP53 c.723del, p.(Cys242Alafs*5).

The profile (in red) was superimposed on the profile of a control individual wild type for TP53 (in blue), using the control amplicons RIC8B and TBP. (C) RT-QMPSF result obtained for the index case’s mother (individual 76, S1. Table 1 and online supplemental table 3) carrying the c.*1175A>C variant at the homozygous state. (D) RT-QMPSF result for the index case (individual 77, online supplemental table 3) carrying the c.723del, p.(Cys242Alafs*5) variant and the c.*1175A>C in trans. Red arrows indicate the appearance of longer p53 transcripts.

The horizontal bars show the reduction of the normal p53 transcript level, as compared with the control. RT-QMPSF, reverse transcription–quantitative multiplex PCR of short fluorescent fragment.DiscussionThe interpretation of germline TP53 variants in patients with cancer is critical and should be performed before starting treatment considering their medical impact. The main objective of our assay was to provide a fast functional classification of rare uncharacterised variants in order to help clinicians with decision-making. Compared with the previous assay that we developed in EBV-immortalised lymphocytes,27 28 this blood assay does not require long-term cell culture and the results can be obtained within 1 week, fulfilling the timing required for diagnostic practice. The only constraint is to perform it within 48 hours after blood sampling in order to obtain robust results.

Under these conditions, we were able to successfully analyse samples sent from other European countries.Our assay fulfils most of the recommendations recently published by the Clinical Genome Resource Sequence Variant Interpretation working group regarding the clinical validity of functional assays29. (1) compared with the previously described p53 functional assays that test in vitro either cloned cDNA in yeast or artificial mutant libraries in cancer cell lines,23–25 this blood assay is performed in clinical samples in the patients’ genetic context. (2) the assay evaluates the transcriptional activity of p53 and not a specific domain of the protein. (3) it analyses simultaneously the impact of the variant on protein function and mRNA levels. (4) it was validated using 51 wild-type TP53 controls and 8 patients with seven distinct pathogenic or likely-pathogenic TP53 variants.

And finally, (5) results show the robustness of the assay. Indeed, as shown in table 1, for 12 tested variants, we were able to perform the assay on EBV-immortalised cell lines and the results were very similar. Moreover, for five individuals, two different blood samples were tested and yielded similar results (table 1), and two variants (c.844C>T, p.(Arg282Trp). C.847C>T, p.(Arg283Cys)) were tested on two different individuals’ blood with concordant results (4.8 vs 5.0 and 5.3 vs 6.4).We observed among the wild-type TP53 individuals a wide range of functionality scores (7.5–22.8). This probably suggests that there is a variability of the p53-mediated transcriptional response to DNA damage in the general population, although no obvious impact of age, clinical status or sex could be observed.

The thresholds used in this study could be refined by testing additional deleterious variants. Despite this variability, all pathogenic/likely pathogenic variants generated low p53 functionality scores, and variants resulting in premature stop codons were also detected by a clear reduction of p53 mRNA levels. In addition, our assay allows testing of non-missense variants such as in frame indels. It should be highlighted that none of the previously published functional assays can be considered as a gold-standard method to classify germline TP53 variants.23–25 Therefore, no available p53 functional assay can be used to calibrate the blood assay. Indeed, as illustrated in table 1, discordant results were obtained for variants unambiguously classified in ClinVar as pathogenic or likely pathogenic.

In particular, the founder Brazilian p.(Arg337His), an example of a variant with low penetrance, highlights the limits of the available tools. Whereas segregation data performed on large Brazilian pedigrees have clearly shown that this variant is pathogenic,34 bioinformatic predictions and functional analyses35 are conflicting (table 1). Our blood functional assay clearly shows that this variant alters the transcriptional activity of p53, although to a lesser extent than DNE missense variations, highlighting the limits of functional assays based on overexpression of cDNA. This result was confirmed in four additional patients carrying this variant using EBV cell lines (table 1).The blood functional assay performed on PBMC harbouring unclassified variants led us to consider 12 variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del)) as ‘functionally abnormal’, some with high impact. The interpretation is particularly challenging for p.(Pro72His), p.(Arg110His), p.(Arg158Cys), p.(Arg283Cys) and p.(Asp352Tyr) variants, as they were considered in yeast assays as functional or partially functional, and the Giacomelli assay classified them as not LOF_not DNE or was not conclusive.

The low functionality score observed for p.(Arg110His) was confirmed in an EBV cell line derived from the patient and confirmed in two EBV cell lines from other patients carrying this variant. The result for the p.(Asp352Tyr) variant was confirmed on a second blood sample and with an EBV cell line derived from another patient also carrying this variant. The effect of p.(Arg283Cys) was also confirmed in EBV cell lines derived from the patient and from three additional patients with the same variant (table 1).The clinical utility of the p53 functional assay is highlighted by the p.(Pro191Arg) variant. This variant was initially detected in a child with medulloblastoma at 2 years of age and whose brother died from a fibrosarcoma. Presymptomatic testing revealed that an unaffected brother (18 months), the mother and two maternal aunts were also carriers.

We were then requested to evaluate this variant, and the functional assay performed in the maternal aunt (individual 65, online supplemental table 3) clearly showed that this variant does not alter the p53 transcriptional activity (table 1 and online supplemental table 3). Considering this result, segregation analysis was performed on the brother’s fibrosarcoma sample, revealing the absence of the variant and consolidating the conclusion of a non-pathogenic variant.Our results show that this blood functional assay is also able to detect TP53 variations outside the coding regions, which are the only regions commonly analysed. Thanks to this assay, we discovered that the unaffected mother of an index case was homozygous for the polymorphic c.*1175A>C variant, and we show that this variant decreases p53 mRNA by altering the polyadenylation signal and produces longer transcripts extending beyond the poly-A site, as previously reported.30 When present on both alleles, this variant impacts p53 functionality with the same magnitude as a germline pathogenic TP53 variant. This prompted us to recommend breast MRI every year for this unaffected adult relative. We had the opportunity to perform the assay on EBV-immortalised lymphocytes harbouring only this heterozygous variant, and we observed a normal score (data not shown), suggesting that the heterozygous c.*1175A>C variant alone is insufficient to alter p53 function.

The comparison of the p53 functional scores observed in the index case who developed a high-grade glioma at 5 years of age and harbours the null c.723del, p.(Cys242Alafs*5) variant and in trans the polymorphic c.*1175A>C variant, and in her father carrying only the TP53 null variant suggests that the c.*1175A>C variant may act as a genetic modifier in pathogenic TP53 variant carriers and could increase the risk of glioma in carriers, as previously shown in the general population.30–33In summary, we suggest that our blood p53 functional assay should be a useful tool not only for the rapid interpretation of germline TP53 variants of unknown significance in clinical practice, in complement to the previously developed assays, but also for the indirect detection of cryptic alterations within regulatory regions impacting p53 function.Data availability statementAll data relevant to the study are included in the article or uploaded as supplementary information. Deidentified participant data are available from thierry.frebourg@chu-rouen.fr.Ethics statementsPatient consent for publicationNot required.AcknowledgmentsThe authors are grateful to their French and European colleagues for providing clinical information and sending blood samples for TP53 analysis. The authors are indebted to Philippe Ruminy (Inserm U1245, Comprehensive Cancer Centre Becquerel, Rouen) for advices on the reverse transcription–multiplex ligation probe amplification experiments and to Nikki Sabourin-Gibbs (Rouen University Hospital) for her assistance in editing the manuscript..