I originally wrote this Post for Penn State WEPO last year but I wanted to share it here for anyone who might want to do some cool things at Penn State or coming to Penn State in the fall. Enjoy! Classes start today!
Hey Ladies! Hope your summer is going great! I know that one of my favorite things to do is to explore towns. see where all the coolest places are, and to try all the food. As a freshman, you probably want to do some cool things with your new awesome friends, so here's a list to help facilitate some awesome memories.
The Library- Penn State's library has the really nifty things called The Stacks where everybody and their whole family tends to congregate during Finals Week but during the semester, it's a good way to study if you like to shut everything out.
The Hub- The Hub is my preferred place of studying. (Say hello if you see me- I'm usually by the Starbucks) It's loud enough where you can get work done and quiet enough that you do not feel like you're sitting in The Cone of Studying Shame.
Otto's Café- In the Keller Graduate Building, there is a café what has comfy chairs, tables and perfect lighting for studying. I really enjoy studying between classes in Keller because you can get a ton of coffee (I really love coffee) and enjoy the light streaming through the window.
The next thing that we love as college students, we love to hang out and make new friends. Sometimes it gets boring to hangout in the same places so here are some nifty places to go.
The Creamery- Some Creamery ice cream or frozen yogurt is a perfect way to talk about trouble with classes, boys and drama. It doesn't hurt that it's a far walk to burn off some calories and it has great salads to take home for dinner if you are like me and eat your dessert before dinner.
The Arboretum- While it's nice out, you should take a walk through the Arboretum. You can really learn a lot about plants and if you go at sunset, the view from the pavilion is gorgeous. It's a really relaxing place to me and perfect place to get to know people.
Kiwi – Downtown State College steals all of my money but Kiwi gets most of it. I love frozen yogurt and I think it's a fun way to catch up with a friend that gets you off campus. I really love the flavor combinations. Last time, I got birthday cake frozen yogurt with sprinkles, chocolate chips, caramel and strawberries. That was delicious!
Chipotle- The haven of all Mexican lovers, Chipotle in State College is a great way to catch up with a large group of people. My friends from PREF and I always go to Chipotle and get these large burritos and bond over the messes we make.
Hub Late Night- This a great thing to do with friends when you want to do something low key and silly. They play movies at like 9 and 12 am, as well as offer deals on food and have fun crafts to make.
Lastly, you should explore the town you live in. There are a ton of things to do in State College that require a little forethought or begging someone to drive, but they are really fun and different than the usual escapades.
State College's Library- This library is not the biggest library I have ever went to but it's a fun play to study as well as read children's books obnoxiously with your friends. You can walk here and it's near Panera Bread for refueling.
Plato's Closet- I had never heard of this store before I came to State College but it's kind of like an upscale thrift shop. It's filled with modern clothes that aren't priced too badly and it's a good place to buy a weird colored shirt for a contest or like the little things you need but do not want to pay the full price. You can take the V bus off campus, and stop and walk to get there.
The Movies- Sometimes you really need to see a movie, especially sequels and crazy awesome new movies like The Hobbit. You can take the M bus out to the movie theatre and then take the M bus back or a taxi. I love doing this with my silliest friends because I just laugh the whole entire time. One taxi ride back, my best friend was singing his new versions of popular songs and it was just a great memory.
Walmart and Dollar Tree- To celebrate people's birthdays, I like to decorate their dorm room door or make them something fun. A trip to Dollar Tree or Walmart is required. These are places that you can find the weirdest things and create some of the sickest crafts with some neat supplies. You can take the V bus out and back for this trip.
I hope that this list can help you have a good time in State College and give you and your friends something to do with you that might be a little different from your usual. I made a little printable that you can keep with you or pin to your bulletin board for inspiration.


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Airport reports banner month for boardings
Published 9:42 am Monday, August 24, 2009
Huntington, W.Va. — For the second straight month more than 10,000 passengers departed from Huntington Tri-State Airport, representing the highest monthly boardings in at least 20 years, according to reports released Thursday during the monthly meeting of the Tri-State Airport Authority.
More than 10,900 travelers departed from the airport in July 2009 — a 31 percent increase compared to the same month in 2008. Year-to-date boardings (62,160) are up 21% compared to the first 7 months of 2008 (51,227).
Allegiant Air, which provides non-stop, low fare service to Ft. Lauderdale, Orlando, St. Petersburg/Clearwater, and Myrtle Beach, carried 62 percent of the airport's passengers in July, and posted a 39 percent increase over July 2008.
The numbers were further boosted by Delta Air Lines, posting a 68 percent increase in boardings over the same month last year. Delta added a third flight to Cincinnati in July, which contributed significantly to the strong increase.
Charlotte service on US Airways remained relatively constant.
Road salt and other de-icers can lead to expensive rust damage to vehicles
Intel building $20B Ohio chip facility amid shortage
FOOD: Tuna Pasta Casserole, Banana Walnut Pancakes
Lifeline of Ohio announces increase in organ, eye, tissue donors
Eight petitions disallowed by BOE

redpajama_set_name: RedPajamaCommonCrawl


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Rep. Stefanik Invites NY-21 Veterans to Share Their Stories
Glens Falls, NY – Today, Congresswoman Elise Stefanik (R-NY-21) announced that she will be sharing stories of North Country veterans on her Congressional website throughout the month of November in honor of Veterans Day.
"The North Country is home to some of the bravest patriots this country has ever known," said Congresswoman Stefanik. "Please help me honor these men and women who have served by allowing me to share their stories and photos with a grateful nation. Our military service members have sacrificed so much to keep our nation safe, and I am honored to serve these brave veterans in Congress."
For residents of New York's 21st district who have served in the military or know someone who has, Congresswoman Stefanik is inviting them to submit photos and stories from their time in service to our office. Congresswoman Stefanik will be adding these stories and photos on her official website (https://stefanik.house.gov/veterans-share-your-story) and Facebook page throughout the month of November.
Please submit your stories and photos to ny21vets@mail.house.gov or to our Glens Falls office at 136 Glen Street, Glens Falls, N.Y., 12801. Please do not mail original photos.
Those who are submitting material are asked to include a short description of the branch, title, and assignment for photos. Also, they are asked to be sure they have permission to share photos and stories before sending.


es; m. A circle or cycle which marks the date. I. the indiction, a cycle of fifteen years. v. ge-ban Ðæm gǽre ðe wæs ágán fram Cristes ácennednesse eahta hand wintra and feówer and sixtig, and in ðam tácencircole ðæt twelfte geár (the year of the indiction is the remainder after dividing 864 + 3 by 15; this remainder is 12, which agrees with the passage ), Chart. Th. 126, 3. II. the lunar cycle of nineteen years; the place which any year occupies in the cycle is marked by the golden number of the year Ðis wæs gewriten on ðam geáre ðe wæs ágán fram Cristes ácennednysse án þusend geára and án and sixtig geára, and an ðam tácncircule ðæt seofanteóðe geár (the golden number of the year 1061 is the remainder after dividing 1061+1 by 19; this remainder is 17, which agrees with the number given in the passage ), Chart. Th. 390, 19.
Bosworth, Joseph. "tácn-circul." An Anglo-Saxon Dictionary: Based on the Manuscript Collections of the Late Joseph Bosworth. Ed. Thomas Northcote Toller. Oxford: Clarendon Press, 1898. 967.


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This is a story about what happens when a video game goes viral by accident, and what happens afterward. • Global aviation authorities want more details from the Federal Aviation Administration about Boeing's proposed fixes for the 737 Max. (NYT)
香 港 红 财 神 六 合 网 二 肖
The 49 state attorneys general who banded together last month in a lawsuit to recover 0 million from one for-profit college company were obviously working under both Republican and Democratic governors. In the face of unquestionable evidence, sometimes bipartisanship isn't so hard. Now it's Congress's turn. When "Beetlejuice" moved into the theater and I walked out onto that stage, I couldn't help but glance up at the house-right side of the mezzanine. I instantly remembered the inverse view, and the little boy whose view it was, unaware of the permanent tattoo being inked into his core. There's a "Memory" joke to be made here, but I'll spare us. After all, I'm sentimental, not cloying. I acknowledge the difference.
芜 湖 中 特 华 星 招 聘
"Lady Chatterley's Lover": The copy of the D.H. Lawrence novel that in 1960 was used by a British judge in one of the most famous obscenity trials of the century was sold at auction last year to an unidentified buyer. Now, the British arts minister is preventing it from being exported until August, as a crowdfunding campaign tries to top the price to keep it in the country. New Yorkers feted him nonetheless. They remembered his near-successes on this site. In 2002, he finished second to Tiger Woods in the United States Open at the Black Course. In 2009, Mickelson tied for second in another U.S. Open. Lucas Glover claimed that trophy, and Mickelson, who has finished second in six U.S. Opens, noted that he continued to feel welcomed by the raucous crowd.
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In revoking her license, the council said she violated the January agreement and "traditional Myanmar culture" by continuing to post revealing images. The euphoria of Woods's inspirational victory at the Masters a month ago was hardly forgotten. But the magic of that moment did not accompany Woods to his next event. Battling an erratic driver — he hit only three of 14 fairways — Woods shot 73 on Friday and missed the cut with a final tournament score of five over.
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Three citizens of Australia are being held in Iran, the Australian government said on Wednesday, confirming the detentions as frictions remain high between Tehran and Western nations over the imperiled 2015 accord limiting its nuclear program. An article on Tuesday about the European Parliament elections misstated the given name of Mr. Macron's parliamentary majority leader. He is Gilles Le Gendre, not Gilbert.
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The protest took a jovial turn from last week, when several students clashed with police, sustained injuries, and were detained and charged with destroying public property. In 1979, however, talks at Lancaster House in London produced a peace deal that ended the bush war, though not the animosities that it had spawned between the rival claimants to the spoils of victory. When Zimbabwe held elections in 2018, Mr. Dabengwa pointedly supported those who opposed Mr. Mugabe's successor.
At least a half-dozen companies that make or distribute prescription opioid painkillers are facing a federal criminal investigation over whether they violated federal law as they shipped more drugs amid the growing nationwide addiction and overdose crisis. "After more than two years of the Trump administration, warnings that trade wars and erratic management style would throw the economy off course have proved wrong so far," Irwin writes, "and tax cuts and deregulation are most likely part of the reason for the strong growth rates in 2018 and the beginning of 2019."
This is Photoshop's version of Lorem Ipsum. Proin gravida nibh vel velit auctor aliquet.Novitec, a well-established German aftermarket firm with a history of modifying Ferrari and Lamborghini models, has more recently been tuning the Tesla Model S and Model X. Now, it's finally turning its gaze to the Elon Musk and Company's most-popular offering, the Model 3. On the visual front, the company's new range of products includes everything from functional aerodynamic enhancements to 21-inch wheels, along with suspension tweaks and cabin refinements. "Now the question is whether the court will be asked to rule on how the constitutional right to equality applies to adoption by married couples of the same sex," she said. "Other issues of concern include whether to legalize surrogacy and changes to laws related to assisted reproductive technology."
The tally was nearly identical to the result of a vote in March, when Congress first sought to block the national emergency declaration and a dozen Republicans joined all Democrats present for the vote. Senator Marco Rubio, Republican of Florida, who voted in March to overturn the emergency declaration, did not vote on Wednesday. Mr. Rubio had a previously scheduled family matter to attend to, but would have voted again to reject the emergency declaration, his office said. "He believes that the pursuit of capitalism is fundamentally moral — that it creates and produces prosperity and progress even when there are immoral actors and even when it creates inequality."
This is Photoshop's version of Lorem Ipsum. Proin gravida nibh vel velit auctor aliquet.Over the years, Lyft has raised .91 billion in private investment funding from firms including Andreessen Horowitz, Founders Fund and China's Didi Chuxing. The fact that it hasn't yet earned a profit might not daunt new investors. The country is already suffering through an unimaginable horror, and watching as a lawless president and a corrupt administration are being allowed to operate carte blanche due to Democrats' squeamishness about their political prospects and compromised conservatives' concerns about their own.
Mayor Sara Duterte-Carpio of Davao ordered the cancellation of all classes in public and private schools, saying the quake "may have caused structural and electrical damage to buildings and houses" in the city. Ms. Duterte-Carpio is the daughter of Rodrigo Duterte, the Philippine president. One of the many ways that Anna Wintour, the Vogue editor and the Met Gala's benevolent dictator, cannily rejiggered what was once an opportunity for society people to flaunt their couture finery is that a hefty percentage of its 550 attendees will be wearing clothes they do not own.
This is Photoshop's version of Lorem Ipsum. Proin gravida nibh vel velit auctor aliquet.CNET's Katie Collins contributed to this report. Over the last three decades, the company, Orsted, has figured out how to build giant wind farms powered by turbines the size of jumbo jets in the shallow waters around northern Europe. These installations generate large streams of clean energy. The electricity they produce also comes at a similar cost to new natural-gas fired plants, according to Deepa Venkateswaran, an analyst at Bernstein, a market research firm. Ms. Venkateswaran said that with many new offshore wind plants expected in the coming years, the cost of building them was likely to continue to fall.
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She appeared in the 1997 romantic comedy "As Good as It Gets" and in Whit Stillman's 1998 film, "The Last Days of Disco," among other movies. Four years ago, she made her directorial debut with "#Horror," about a rich-girl sleepover gone awry, a film that was coolly received. He grew up in a crime-ridden pocket of the San Fernando Valley, in northwestern Los Angeles, the oldest of six children raised by a single mother who heckled neighborhood drug dealers. Bray went on to a successful career as a Hollywood stuntman — his credits include "Iron Man" and "Star Trek" — and then a television director. But his youngest brother, Craig, got caught up in gang life and died.
Square Enix promised to reveal more about the release on Monday at the company's E3 press event. The first question that comes to mind about the release date is whether the entire game will be available on March 3. Producer Yoshinori Kitase previously said the game would be released in multiple Final Fantasy XIII-sized installments. The proposals from Ms. Harris and Mr. Booker, and one of Mr. Brown's plans, would deliver larger benefits for taxpayers in the middle of the income distribution than Mr. Trump's cuts did, the analysis found. The less expensive of Mr. Brown's plans would deliver smaller benefits, as would Mr. Bennet's, though both plans would give a larger share of their overall windfalls to the middle class than Mr. Trump's did.
Mr. Bel is not the only internationally successful artist with an anti-flying stance. The Berlin-based performance artist Tino Sehgal refuses to travel by plane, as does the British theater director Katie Mitchell. (There are even fewer cultural institutions with this policy; the Helsingborg Concert Hall in Sweden is a rare example.) — James Tapscott
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Gov. Andrew M. Cuomo has confirmed he will sign the measure into law. He said he tried using antibiotics for one season, but gave up after seeing little improvement. Cost, he added, was the main reason he didn't continue spraying.
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The Mini Crossword: Here is today's puzzle. The women's dancing is bewitching in another way. "It's elegant, it's fluid, it's flowing," Mr. Salaam said. "It's a living sense of grace. This is another construct of beauty and elegance." He paused to sigh. "The litheness of it."


text:
Potentilla ulrichii är en rosväxtart som beskrevs av Gerald Parolly och Nordt. Potentilla ulrichii ingår i Fingerörtssläktet som ingår i familjen rosväxter. Inga underarter finns listade i Catalogue of Life.

Källor

Fingerörter
ulrichii

redpajama_set_name: RedPajamaWikipedia


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Марсія́к () — колишній муніципалітет у Франції, у регіоні Нова Аквітанія, департамент Жиронда. Населення —  осіб (2011).

Муніципалітет був розташований на відстані близько 460 км на південний захід від Парижа, 50 км на північ від Бордо.

Історія

1 січня 2019 року Марсіяк і Сен-Капре-де-Бле було об'єднано в новий муніципалітет Валь-де-Лівенн.

Демографія

Розподіл населення за віком та статтю (2006):

Економіка

У 2010 році в муніципалітеті числилось 496 оподаткованих домогосподарств, у яких проживали 1174,5 особи, медіана доходів виносила  євро на одного особоспоживача

Сусідні муніципалітети

Посилання

 Марсіяк на сайті французького Національного інституту географії
 [ Розташування муніципалітету Марсіяк на мапі Франції та сусідні муніципалітети]

Див. також
 Список муніципалітетів департаменту Жиронда

Примітки

Колишні муніципалітети департаменту Жиронда


Why Does This President Intentionally Delay Submission of a Budget?
From the time Obama assumed the office of POTUS, he has disobeyed the law regarding submitting a #budget.
It's now six years into his reign of economic #plunder and he again flaunts the law. He is required by law to submit a budget to Congress no later than the first Monday in February. Six times late. Why?
Obama's ploy is and has been to intentionally obfuscate expenditures from us.
Barack Obama, empowered by a Democrat Senate majority, has added more national debt than the sum of all previous presidents from the first "George" to the last "George".
As of this morning, 3/6/014, national debt has skyrocketed to the astronomical sum of $17,409,592,000,000! We are running a deficit over $638 billion. Those of us who pay taxes – we are called taxpayers – owe $150,827 EACH – JUST TO SATISFY NATIONAL DEBT.
Year by year, the Congressional Budget Office warns, as it does this year, that Obama's spendthrift ways are unsustainable. What does unsustainable mean?
Unsustainable means "incapable of being maintained at a steady level". Let's try a different definition: it means we are headed into the most disastrous bankruptcy the world has ever known.
Are you listening? Do you understand? If yes, can you share your understanding with everyone you know?
Tell Congress to cut federal spending on agencies that in and of themselves threaten our national existence: Environmental Protection Agency (all states have their own), Dept. of Education (all states have their own), Dept. of Energy (created to help, it now works against us), Dept. of Homeland Security, aka Obama's National Police Force (you know the drill) and all funding to nations who wish us evil.
Keynesian economists believe taxes and revenue run parallel paths: increasing taxes will increase revenue. Presidents Kennedy and Reagan disproved this false economic theory.
Foremost in endangering America is the Affordable Care Act, aka ObamaCare. Tell Congress you want #FullRepeal. This law is utterly destroying the medical insurance industry and it is causing closures of regional hospitals.


i came across these super cool iphone cases from sonix on instagram. last week when i was in bangkok i found some cool iphone cases and i'm a sucker for all kinds of decorative gadgets. check them out here!


In today's rapidly evolving IT environment, trends like workplace mobility, IT consumerization and new technologies like Cloud, are driving a significant shift in the way IT is being consumed and delivered. We believe 2013 will see more CIOs and IT execs completely rethinking the traditional outsourcing relationship, starting with the contract. More deals will be struck that allow for contingencies and variable conditions. And there will be more transparency and alignment between the service provider and the customer. The five-year fixed, "black-box" contract will be an artifact of the past.
This case study is from the series, "The End of Traditional Outsourcing". The series is HCL's attempt to showcase North America's biggest second generation outsourcers in new age engagements, that scope for future technology and innovations that improve business performance.
This is the story of a global leader in embedded processing solutions, providing industry-leading products that are advancing the automotive, consumer, industrial and networking markets.


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Blasphemous Collector's Edition - Limited Run Games - (Playstation 4)
LRG-PS4-BCI
A foul curse has fallen upon the land of Cvstodia and all its inhabitants - it is simply known as The Miracle.
Play as The Penitent One - a sole survivor of the massacre of the 'Silent Sorrow'. Trapped in an endless cycle of death and rebirth, it's down to you to free the world from this terrible fate and reach the origin of your anguish.
Explore this nightmarish world of twisted religion and discover its many secrets hidden deep inside. Use devastating combos and brutal executions to smite the hordes of grotesque monsters and titanic bosses who are all ready to rip your limbs off. Locate and equip relics, rosary beads and prayers that call on the powers of the heavens to aid you in your quest to break your eternal damnation.
Blasphemous for the PlayStation 4.
Original soundtrack.
Double sided 18" x 24" poster.
Metal pin.
Fine art print.
The art of Blasphemous book.
5" Mea Culpa sword replica.
Title: Blasphemous Collector's Edition
Type: Action
Region Code: USA
Publisher: Limited Run Games
Blasphemous Collector's Edition - Limited Run Games for Playstation 4 is available at VideoGamesNewYork, NY
Blasphemous Collector's Edition - Limited Run Games - (Nintendo Switch)
A foul curse has fallen upon the land of Cvstodia and all its inhabitants - it is simply known as The Miracle.Play as The Penitent One - a sole survivor of the massacre of the 'Silent...
Dark Devotion - Limited Run Games (Playstation 4)
Dark Devotion - Limited Run Games - (Playstation 4) Dark Devotion features a richly detailed, immersive narrative that unfolds a sombre tale of your Templar religion's irresistible calling...
Sephirothic Stories - Limited Run Games - (Playstation 4)
Sephirothic Stories - Limited Run Games - (Playstation 4) When the world tree begins to wane, what will become of the balance of the world? Discover the truth behind the world tree's waning power in...
VA-11 HALL-A Collector's Edition - Limited Run Games (PlayStation 4)
VA-11 HALL-A Collector's Edition - Limited Run Games (PlayStation 4) In this world, corporations reign supreme, all human life is infected with nanomachines designed to oppress them, and the...
Blasphemous - Limited Run Games - (Playstation 4)
Blasphemous is a punishing action-platformer that combines the fast-paced, skilled combat of a hack-n-slash game with a deep and evocative narrative core, delivered through exploration of a huge...
Blasphemous LR-52 (Nintendo Switch)
Blasphemous LR-52 for Nintendo Switch Blasphemous is a punishing action-platformer that combines the fast-paced, skilled combat of a hack-n-slash game with a deep and evocative narrative core,...
Blossom Tales: The Sleeping King - Limited Run Games - (Nintendo Switch)
Explore an enchanted world in classic action-adventure fashion. Explore a vast open game world in classic action-adventure fashion as Lily, Knight of the Rose!Slash your way through...
ADK Damashii Classic Edition - Limited Run Games (Playstation 4)
ADK Damashii
ADK Damashii Classic Edition - Limited Run Games - (Playstation 4) Ganggang Kohshinkyoku (abbreviated ADK) is a one-on-one fighting arcade game that includes the ability to fight on multiple planes...
Dark Devotion - Limited Run Games - (Nintendo Switch)
Explore the secrets of a mysterious fallen temple and put your Templar faith to the test in Dark Devotion, where no sacrifice is too great in praise of your God. Measure your devotion and your...
River City Girls (Nintendo Switch)
River City Girls for Nintendo Switch from Limited Run Games There's trouble once again on the mean streets of River City, but this time the boys are in over their heads! In this all-new entry in the...
Grandia HD collection- Limited Run (Nintendo Switch)
Grandia HD collection- Limited Run (Nintendo Switch) GRANDIA tells the story of Justin, the protagonist, and his companions in a race against the evil Garlyle Forces to uncover the long-lost secrets...
VA-11 HALL-A - Limited Run Games - (Playstation 4)
VA-11 HALL-A - Limited Run Games - (Playstation 4) In this world, corporations reign supreme, all human life is infected with nanomachines designed to oppress them, and the terrifying...


The 8 cars that took top honours at the 2015 Tow Car Awards across each category.
2015 Tow Car Awards winners include 8 categories of car and an overall champion. Each excelled in a series of challenging tests such as the: high speed stability, emergency stop, lane changing and hill-start – all while coupled to a caravan or weighted trailer. Running costs, practicality and solo performance played a role too. The winners were chosen by a panel of judges from Practical Caravan, the Camping and Caravanning Club and What Car?
The ŠKODA Octavia Hatchback claimed the Up to 1,400kg title (2.0 TDI CR 150PS SE Business trim). Judges said – in relation to the lane change test - that "on the third and fastest run the caravan was sliding dramatically, but the ŠKODA pulled it straight again without being dragged off its line." The Octavia also won praise for its engine that enabled it to proceed at a "decent pace". It also took the hill-start test "in its stride" and was "immensely roomy". Experts said it was "hard to find any significant weakness".
The Volkswagen Passat Estate won the 1,400 to 1,549kg title (2.0 TDI BMT 150PS SE Business DSG). It took the overall crown too. Judges said it punched "above its weight with a caravan in tow" and felt "rock solid" at 60mph. As for changing lane, it made the "most aggressive test seem easy cornering hard and bullying the caravan into following", the judges added. The Passat Estate was also praised for its "very roomy" cabin, "huge" cargo capacity and electric parking brake that made "short work" of its hill-start test.
The Mazda CX-5 took top spot in the 1,550 to 1,699kg category (2.2D 175PS AWD Sport Nav). Judges said its mid-life update "rounded off a couple of rough edges and made the CX-5 into a really superb tow car." It was applauded for feeling "completely settled at 60mph" and being "very nearly as comfortable at 70mph". The large boot and reversing camera found favour too. The latter was "a great help when hitching up", experts added. Finally, even when "thrown around" it took the "punishment and felt like it could cope with more".
The VW Passat Estate won the 1,700 to 1,899kg category too (albeit in 2.0 BiTDI SCR BMT 240PS 4Motion GT DSG form). Strengths included its diesel engine that delivered "the punch of a heavyweight boxer". It had 237bhp and 369lb ft of torque. This complemented a 4-wheel-drive system and electronic handbrake that ensured "effortless hill starts", judges said. Furthermore, when slowing from speed there was "no instability from the caravan". It was "just as competent swerving hard as driving a straight road at" at 60mph.
The Land Rover Discovery took the 1,900kg+ title (3.0 SDV6 SE Tech). Experts said it felt "absolutely superb" thanks – in part – to its "thoroughly sorted suspension". Straight-line stability was "excellent" and "you'll need to be going well beyond the legal limit or driving in a strong crosswind before you need to make any steering corrections". The lane change was "so easy". The engine and brakes were "top-notch" too, as was the hill start. It also scored well for practicality thanks to its 7 seats, vast boot and the wide range of towing aids.
The Volvo V40 won the Green Award title (Cross Country D4 Lux Nav Geartronic). Judges said it was "very impressive" that a car of this size, weight and performance averages 65.7mpg and meets Euro 6 emission standards. The V40 also found favour for its hill-start and engine that ensured it felt quick and decisive". There was "no instability when slowing from high speeds", and "it made the lane-change test seem easy". Judges said "even when the caravan began to move around the Cross Country remained firmly in control".
The Ford Mondeo 1.5T EcoBoost claimed the Best Petrol Tow Car title. Judges said the engine was "a star" and – even though it lacked pulling power compared to the diesel equivalent - its "177lb ft torque made it "noticeably stronger in the middle of the rev range than most petrols". The Mondeo excelled in other areas too. "The lane-change test held no fear", it pulled the caravan straight when it wandered at speed, and the big boot made packing easy. Judges were pleased to note the presence of a trailer stability system.
The Citroen C4 Cactus claimed the 2015 Best Ultralight Tow Car title. The judges explained that it is particularly suitable for towing a trailer tent and for those that do not want, or need, a large vehicle. Naturally, stability is a prime consideration for towing and the Citroen "pressed home its advantage" over its competitors. During the lane change test it "felt secure" and whereas the trailer" slid around on the third and fastest run", it "knuckled down and dragged it straight" The C4 "stopped well too".


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Morphologic and molecular correlates of EZH2 as a predictor of platinum resistance in high-grade ovarian serous carcinoma
Brett M. Reid1,
Shraddha Vyas1,
Zhihua Chen2,
Ann Chen2,
Peter A. Kanetsky1,
Jennifer B. Permuth1,
Thomas A. Sellers3 na1 &
Ozlen Saglam4 na1
Enhancer of zesta homologue 2 (EZH2) is an essential component of polycomb repressive complex 2 (PRC2) that contributes to tumor progression and chemo-resistance. The aim of this study was to comprehensively assess the prognostic value of EZH2 across the morphologic and molecular spectra of high-grade serous ovarian carcinoma (HGSOC) by utilizing both immunohistochemistry (IHC) and proteogenomic technologies.
IHC of EZH2 was performed using a tissue microarray of 79 HGSOC scored (+/−) for lymphovascular invasion (LVI), tumor-infiltrating lymphocytic aggregates ≥1 mm (TIL) and architectural growth patterns. The association of EZH2 H-score with response to therapy and overall survival was evaluated by tumor features. We also evaluated EZH2 transcriptional (RNA sequencing) and protein (mass spectrometry) expression from bulk tumor samples from 336 HGSOC from The Cancer Genome Atlas (TCGA). EZH2 expression and co-expression networks were compared by clinical outcomes.
For HGSOC without TIL (58%), EZH2 expression was almost 2-fold higher in platinum resistant tumors (P = 0.01). Conversely, EZH2 was not associated with platinum resistance among TIL+ HGSOC (P = 0.41). EZH2 expression was associated with reduced survival for tumors with LVI (P = 0.04). Analysis of TCGA found higher EZH2 expression in immunoreactive and proliferative tumors (P = 6.7 × 10− 5) although protein levels were similar across molecular subtypes (P = 0.52). Both mRNA and protein levels of EZH2 were lower in platinum resistant tumors although they were not associated with survival. Co-expression analysis revealed EZH2 networks totaling 1049 mRNA and 448 proteins that were exclusive to platinum sensitive or resistant tumors. The EZH2 network in resistant HGSOC included CARM1 which was positively correlated with EZH2 at both mRNA (r = 0.33, p = 0.003) and protein (r = 0.14, P = 0.01) levels. Further, EZH2 co-expression with CARM1 corresponded to a decreased prognostic significance of EZH2 expression in resistant tumors.
Our findings demonstrate that EZH2 expression varies based on its interactions with immunologic pathways and tumor microenvironment, impacting the prognostic interpretation. The association between high EZH2 expression and platinum resistance in TIL- HGSOC warrants further study of the implications for therapeutic strategies.
Epithelial ovarian cancers (EOCs) make up the fourth leading cause of cancer related deaths for women between ages 40–59 [1]. Since the disease is typically diagnosed at advanced stages, survival remains poor, with little improvement since the introduction of platinum-based treatment almost 40 years ago [2]. High-grade serous ovarian carcinoma (HGSOC) is the most common (60%) histotype and also very aggressive, representing more than 70% of all EOC deaths [3]. Standard first-line treatment is surgical cytoreduction combined with platinum-based chemotherapy. Although several new and promising agents have been investigated in recent years, systemic or intraperitoneal platinum-based cytotoxic chemotherapy remains the standard initial approach to advanced and recurrent ovarian cancer. While the majority of HGSOC tumors are optimally debulked and display initial sensitivity to platinum-based regimens, most ultimately recur and become refractory to treatment [4].
Enhancer of Zesta Homologue 2 (EZH2) is the catalytic unit of Polycomb Repressive Complex 2 (PRC2) which functions as a highly conserved histone methyltransferase targeting H3K27 to induce target gene silencing [5]. EZH2 is involved in the regulation of cell cycle progression and its dysregulation results in accelerated cell proliferation and cancer development. Additionally, EZH2 contributes to tumor invasion and metastasis through the modulation of angiogenesis and the epithelial-to-mesenchymal transition [6]. Overexpression of EZH2 has been observed in a wide variety of cancers including ovarian cancer where its upregulation has been shown to occur with acquired cisplatin resistance in ovarian cancer cell lines and xenografts [7]. Conversely, knockdown of EZH2 re-sensitizes drug-resistant ovarian cancer cells to cisplatin [7].
EZH2 overexpression has been associated with poor survival in breast [8], prostate [9], and colorectal cancers [10], and studies for EOC have yielded similar results [11, 12]. EOC studies have found that EZH2 expression is upregulated with advancing stage and grade of disease, and among HGSOC with lymph node involvement [11, 13]. However, the prognostic significance of EZH2 has not been evaluated across the diverse morphologic and molecular features of HGSOC. Studies have shown tumor features such as tumor-infiltrating lymphocytes [14] and architecture pattern [15, 16] correspond with favorable prognosis and chemosensitivity. Furthermore, HGSOC have been differentiated into at least four molecular subtypes with differential patterns of gene expression [17, 18]. We therefore questioned whether associations between EZH2 expression and clinical outcomes varied by morphologic or molecular features of HGSOC. We performed an immunohistochemical analysis of EZH2 protein expression in a tissue microarray (TMA) of HGSOC with detailed morphological assessment. In addition, we used The Cancer Genome Atlas (TCGA) data to explore EZH2 mRNA and protein expression among molecular subtypes of HGSOC and the associations with prognosis. Finally, we performed co-expression analysis to identify differential co-expression networks underlying the prognostic relationship.
Study participants and clinical data
This study included data and specimens from women diagnosed with pathologically-confirmed HGSOC at Moffitt Cancer Center and Research Institute (Tampa, Florida). All women received cytoreductive surgery and subsequent first-line platinum-based chemotherapy and were followed for response to therapy (RTT). Participants provided written informed consent for research use of data and biospecimens through Total Cancer Care [19] protocol and usage for this study (#Pro00026247) was approved by the IRB of the University of South Florida. Demographic, clinical, and pathologic data were obtained from the Moffitt Cancer Registry (pathological diagnosis, degree of debulking/cytoreduction, histology, stage, grade), National Death Index (vital status), and medical record abstraction (chemotherapy regimens and response, BRCA status). Patient response to therapy was classified as platinum sensitive for complete responders with disappearance of all measurable disease, or in the absence of measurable lesions, a normalization of CA-125 level for 4 weeks, based on established guidelines [20]. Incomplete responders with partial response (defined as a 50% or greater reduction in tumor burden obtained from measurement of each bi-dimensional lesion for at least 4 weeks, or a drop in CA-125 by > 50% for at least 4 weeks), stable disease, or progression were considered platinum resistant.
Tissue microarray and immunohistochemistry
We constructed a tissue microarray (TMA) of formalin-fixed paraffin embedded primary neoplastic tissue and unaffected benign ovarian tissues. Duplicate 1.0 mm cores were sampled to account for tissue heterogeneity. In total there were 96 HGSOC subjects, 29 of which had paired adjacent normal ovarian epithelium, non-neoplastic stroma, and/or fallopian epithelium sampled across five TMA slides. Slides were stained using a Ventana Discovery XT automated system (Ventana Medical Systems, Tucson, AZ) as per manufacturer's protocol with proprietary reagents. Briefly, after deparaffinization (EZ Prep), heat-induced antigen retrieval was performed (standard CC1; Tris-EDTA buffer pH 7.8 at 95 °C for 44 min). Slides were incubated with rabbit anti-EZH2 (#790–4651) at predilute concentration followed by anti-rabbit antibody (OmniMap anti-Rt HRP, 760–4311). Slides were then immunostained with diaminobenzidine (ChromoMap DAB kit, 760–159) and counterstained with Hematoxylin. Negative control slides omitting the primary antibody were included in all assays.
EZH2 nuclear expression was manually reviewed and scored using histoscore (H-score; range 0–300) that was calculated by multiplying the percentage of positively stained cells by nuclear staining intensity (1+, 2+, 3+). Readings from multiple cores were averaged. IHC stained slides were also imaged with a Leica Aperio AT2 digital pathology scanner (Leica Biosystems, Vista, California) through a 20X/0.7NA objective lens. The percentage of positively stained nuclei was determined using Aperio's Nuclear algorithm with default settings. Nuclei were binned by stain intensity (0, 1+, 2+, 3+) and digital H-score was calculated.
Pathology review
Comprehensive pathologic review of all available H&E slides was performed to annotate morphological features. Histological subtypes other than HGSOC (e.g. mixed carcinomas and low-grade serous carcinomas) were excluded from further analysis (n = 17). For tumor grading, a previously described binary system was used [21]. Morphologic features were evaluated in the primary and metastatic anatomical sites with an average of 22 slides reviewed per case. Papillary architecture was defined as tumor cells arranged around finger-like vascular connective tissue. Micropapillary architecture displayed dyshesive tumor cell clusters lacking a central vascular core surrounded by retraction space. Solid architecture was defined as tumor cells arranged in sheets. Pseudoendometrioid/pseudoglandular architecture showed tumor growing in back-to-back nested pattern with punched out pseudo lumens.
Primary statistical analyses of HGSOC EZH2 expression analyzed manual H-score continuous measurements. EZH2 scores were compared between groups using the Wilcoxon rank-sum test. The association of clinicopathologic and morphological characteristics and EZH2 expression with response to therapy (sensitive/resistant) were evaluated using logistic regression models. Associations with overall survival (hazard ratio, HR) were estimated using cox proportional hazards models. Adjusted models included significant clinicopathologic factors (P < 0.05) using forward stepwise selection with P < 0.10 as entrance criteria. Survival associations were additionally assessed for effect modification by entering interaction terms for EZH2 and morphological variables into the models, and stratified analyses were performed when significant (P < 0.05). As a sensitivity analysis, we secondarily performed all association analyses using digital EZH2 H-score to assess robustness of statistical findings to the method of EZH2 quantification. Statistical analyses were performed in SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).
Gene expression analysis in The Cancer Genome Atlas (TCGA)
We obtained molecular and clinical data for HGSOC samples from The Cancer Genome Atlas (TCGA). Clinical data were downloaded from the Genomic Data Commons data portal (https://portal.gdc.cancer.gov/) and molecular subtypes were annotated using classifications from the original TCGA mRNA expression analysis [17]. Level 1 (FASTQ) RNA sequencing data was downloaded using the gdc-client tool and aligned to the human reference genome build 37 (hs37d5) using tophat (version 2.0.13) and bowtie2, and gene-level reads was quantified based on GENCODE v25 gene model using HTSeq (version 0.6.1). The R package DESEQ2 was used to generate Fragments Per Kilobase of transcript per Million (FPKM) values from the raw expression counts. A total of 60,252 genes were quantified and analyses were limited to 336 HGSOC subjects with complete clinical data. Normalized relative protein expression was downloaded for 172 TCGA HGSOC subjects characterized by the Clinical Proteomic Tumor Analysis Consortium [22] and 8154 proteins quantified in at least 30% (n > 51) of subjects were included in the analysis. Tumor immune cytolytic activity metric was calculated as the geometric mean of GZMA and PRF1 (mRNA) expression which correlates with tumor T cell activation and interferon-stimulated chemokines (CXCL9, CXCL10) [23].
We performed linear regression to test for differences in continuous (log2 transformed) EZH2 expression by clinicopathologic factors, and logistic regression to estimate the association with RTT (resistant/sensitive). Cox proportional hazard modeling was used for survival analyses. All models evaluating EZH2 expression were adjusted for tumor purity using the consensus purity estimate [24]. Covariate adjustment for age, stage, residual disease, and molecular subtype were tested for significance (P < 0.10). Residual disease and subtype were included in RTT models while age and residual disease were included in survival models.
Co-expression analysis was limited to 31,238 genes with median expression >30th percentile. Gene expression correlations were calculated separately for mRNA and protein datasets using the partial correlation between EZH2 and each gene, controlling for tumor purity. Partial pearson correlations were calculated using log2 transformed mRNA expression levels. Protein expression levels were already normalized as previously described [22]. Differential co-expression analysis to statistically compare differences in EZH2 correlations by RTT and perform gene ontology enrichment analyses for differentially correlated genes was performed using the R package DGCA [25]. Briefly, DGCA computes and transforms correlation coefficients to z-scores and uses differences in z-scores to calculate empirical p-values of differential correlation between genes using permutation testing. We modified the DGCA pipeline to compare partial correlations by inputting residual expression values after the effect of tumor purity was regressed out. The DGCA wrapper function to perform gene ontology (GO) enrichment analysis is based on the GOstats R package (version 2.52) [26] and org. Hs.eg.db GO annotation R package (version 3.10.0). To visualize the differentially expressed EZH2 co-expression networks, we used the MEGENA R package (version 1.3.7) [27] to build a planar filtered network from differentially correlated gene pairs (P < 0.05) and identify hierarchal gene modules in the network using default parameters. We then constructed a graph of the gene expression correlation modules using Cytoscape (version 3.8.2) [28]. Statistical analyses were performed using R version 3.6.2 software (www.r-project.org).
Clinicopathologic characteristics
Clinical and pathological characteristics of the 79 study participants with pathologically confirmed HGSOC samples are summarized in Table 1. The median age at diagnosis was 61 years and most (90%) were diagnosed at an advanced stage. Median survival was 4.1 years and the majority (75%) were platinum sensitive. Optimal debulking status (residual disease < 1 cm) was achieved during cytoreductive surgery for most patients and was similar between platinum sensitive and resistant cases (79% vs. 74%, respectively). Out of 21 cases with known BRCA germline mutation status, 11 were BRCA1 or BRCA2 mutation carriers. Carriers were significantly younger than non-carriers (49 years vs. 61 years, P = 0.01), but were otherwise similar across clinicopathologic characteristics and response to therapy (RTT) (see Table S1, Additional file 1).
Table 1 Clinicopathologic characteristics of study participants
Morphologic features assessed included presence of cystic component, lymphovascular invasion (LVI), serous tubal intraepithelial carcinoma (STIC; Fig. 1A), and intratumoral lymphoid aggregates within the peritumoral stroma measuring 1 mm or larger in size (tumor-infiltrating lymphocytes or TIL; Fig. 1B). Most tumors presented with LVI (79%) and cystic component (69%) features. LVI was less common among early stage cases than advanced stages (25% early vs. 85% advanced, P = 0.0009). TIL measuring 1 mm or larger were present in 41% of patient cases and did not vary by stage (P = 0.59). None of the pathologic features were associated with residual disease or RTT.
Representative slide images for (a) serous tubal intraepithelial carcinoma (STIC), (b) tumor-infiltrating lymphocytes (TIL), and tumor architectural growth patterns: (c) papillary, (d) micropapillary, (e) solid, and (f) pseudoglandular. Original magnification: 400x for a, d, e and 200x for b, c, f
Tumor architectural growth patterns were characterized for all subjects which included papillary, solid, pseudoendometrioid/pseudoglandular, and micropapillary patterns (Fig. 1C-F). Patterns were reviewed across clinicopathologic factors (Table 2). The most predominant architectural patterns were papillary (76%) and pseudoglandular (76%) followed by solid (71%) and micropapillary (62%). Tumors often displayed multiple morphological features with most displaying three (50%) or all four patterns (22%) (see Table S2, Additional file 1). Micropapillary pattern was more common in subjects with advanced disease stage (P = 0.0002) and LVI (P < 0.0001). Papillary pattern was more common when a cystic component was present (P = 0.04). Subjects with STIC were more likely to have pseudoglandular pattern (P = 0.03). None of the architectural patterns or combination of patterns observed were associated with RTT.
Table 2 Tumor architectural growth patterns by clinicopathologic characteristics
Clinical factors associated with overall survival included age at diagnosis (HR = 1.06, 95% CI = 1.03–1.09, P = 0.0001), advanced stage of disease (HR = 9.02, 95% CI = 1.24–65.44, P = 0.03), and platinum sensitivity (HR = 0.30, 95% CI = 0.16–0.58, P = 0.0003). After adjusting for these clinical factors, LVI and the papillary pattern were independently associated with poor survival (Table 3; HR = 3.36, P = 0.007 and HR = 2.39, P = 0.02, respectively). In stratified analysis, the association of papillary pattern with reduced survival was only observed in cases without a cystic component (P interaction = 0.02) in whom over a 5-fold higher hazard of death was observed when a papillary pattern was present (HR = 5.6, 95% CI = 1.5–20.4, P = 0.01). Further, the solid architectural pattern was associated with improved survival (HR = 0.24, 95% CI = 0.07–0.85, P = 0.03) but only in TIL- cases (P interaction = 0.004).
Table 3 Association of tumor morphological features with overall survival
EZH2 expression and clinicopathologic features in HGSOC
EZH2 expression was evaluated in tumor epithelium of all subjects included in the study. Representative immunohistochemical staining of EZH2 is shown in Fig. 2A. Median H-score of EZH2 was 140 with a range of 10 to 270 in neoplastic tissue (Fig. 2B). For 24 cases with paired normal fallopian tube tissue, we observed significant overexpression of EZH2 within the neoplastic tissue (P = 4.4 × 10− 10; Fig. 2C and D). Five cases demonstrated similar overexpression of EZH2 in HGSOC compared to paired normal ovarian epithelium (P = 0.04, Fig. 2D). EZH2 expression was absent from normal stroma samples (n = 40) and was only quantified digitally.
(a) Immunohistochemical (IHC) expression of EZH2 in low, median, and high expressing HGSOC samples at 5x and 20x magnification. (b) Distribution of EZH2 H-score in HGSOC tissues. (c) IHC expression of EZH2 in paired HGSOC and adjacent normal fallopian tube (FT) tissue in representative subject. (d) Boxplots of EZH2 H-score in HGSOC (n = 27) and paired normal epithelium (n = 5) and normal fallopian tube (FT, n = 24) tissues. Groups were compared using the paired t-test. (e) Boxplots of EZH2 H-score in HGSOC and normal appearing FT tissues for subjects with STIC (n = 20) and without STIC (n = 51). Groups were compared using the Wilcoxon two-sample test. (f) Boxplots of HGSOC EZH2 H-score grouped by response to therapy (sensitive/resistant) and presence/absence of TIL. The symbols in the boxplots indicate the mean for (o) resistant and (+) sensitive subgroups. Horizontal line in box interior represents the group median. Groups were compared using the Wilcoxon two-sample test. (g) Adjusted survival curve estimates for EZH2 expression for the lowest quartile (Q1) to the highest quartile (Q4) using cox proportional hazards regression. Adjustments include age, stage, and response to therapy. Statistical significance of boxplot comparisons is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.0001
Tumor EZH2 expression was slightly higher for cases with suboptimal debulking status (P = 0.06) and platinum resistance (P = 0.09), but it was not associated with disease stage (P = 0.92) or age of diagnosis (P = 0.23) (Fig. S1, Additional file 1). Overexpression of EZH2 was observed in HGSOC tissue from cases that were confirmed to have STIC present (n = 20) compared to HGSOC from cases without STIC (n = 51, P = 0.01) whereas expression did not differ in normal appearing fallopian tube tissue obtained from HGSOC cases with STIC (n = 5) compared to those without STIC (n = 14, P = 0.37, Fig. 2E). EZH2 was not differentially expressed based on other morphological characteristics or BRCA mutation status (P = 0.76).
EZH2 expression and HGSOC prognosis
Among tumors without TIL, EZH2 upregulation was associated with platinum resistance (OR = 1.03, 95% CI = 1.01–1.05, P = 0.01; Fig. 2F); however, no association (OR = 0.99, 95% CI = 0.98–1.01, P = 0.41) was observed for tumors with TIL present (P interaction = 0.01). EZH2 was not significantly associated with RTT for any other morphologic subgroup. Expression of EZH2 was not associated with overall survival among all HGSOC (P = 0.79). When stratified by morphological characteristics, increasing EZH2 expression was nominally associated with reduced survival (HR = 1.00, 95% CI = 1.00–1.02, P = 0.04) among 62 cases with LVI present (P interaction = 0.02). For HGSOC with LVI, mean survival was 32 months in the top quartile of EZH2 expression compared to 42 months in the bottom quartile (Fig. 2G). This effect was slightly more pronounced in the majority (76%) of LVI cases with micropapillary pattern (HR = 1.01, 95% CI = 1.00–1.02, P = 0.02). For HGSOC without LVI, there was no association observed between EZH2 expression and overall survival (HR = 0.99, 95% CI = 0.98 to 1.00, P = 0.10).
Digitally quantified EZH2 H-scores ranged from 3 to 126 (median = 28) and were highly correlated to manually obtained H-scores (spearman r = 0.80, P < 0.001). Secondary analyses using digital H-score replicated our primary results for EZH2 upregulation in HGSOC tissues compared to paired fallopian tube (P = 2.8 × 10− 5). Digital quantification of EZH2 in normal stroma confirmed expression was largely not detected (Fig. S2, Additional file 1). Higher EZH2 expression was observed in patients with suboptimal debulking (P = 0.02), but differential expression was not observed for response to therapy (P = 0.50) or presence of STIC (P = 0.14). By morphological features, we observed that higher EZH2 expression in HGSOC without TIL was associated with platinum resistance (OR = 1.04, 95% CI = 1.01–1.07, P = 0.02) using digital H-score. EZH2 expression was associated with reduced survival (HR = 1.01, 95% CI = 1.00–1.03, P = 0.03) for HGSOC with LVI, but micropapillary pattern was not significant (P = 0.06).
EZH2 mRNA and protein expression among HGSOC molecular subtypes in TCGA
To understand the dynamics of EZH2, we explored transcriptome and proteomic co-expression networks across the clinicopathologic and molecular features of 336 clinically characterized HGSOC in TCGA. EZH2 transcript levels were correlated with protein levels in 72 cases with both mRNA and protein measurements (r = 0.29, P = 0.01). EZH2 mRNA expression differed across the four molecular subtypes of HGSOC [17] exhibiting higher expression in immunoreactive and proliferative tumors (P = 6.7 × 10− 5); however, EZH2 protein levels did not differ (P = 0.52, Fig. 3A). Given the importance of TIL in our IHC analysis of EZH2, we evaluated EZH2 mRNA expression by cytolytic activity score, which correlates with T cell infiltration [29]. Cytolytic activity varied by molecular subtype (P = 2.2 × 10− 16) with immunoreactive tumors showing the highest cytolytic activity metric followed by mesenchymal, differentiated, and proliferative (Fig. 3A). EZH2 mRNA and protein levels did not correlate with cytolytic activity metric (mRNA r = 0.08, P = 0.15; protein r = − 0.16, P = 0.17). We also assessed EZH2 expression by molecularly characterized tumor-immune phenotypes. Tumors were classified as immune deserts (CD8+ T cells are either absent or present in very low numbers), immune excluded (infiltrating CD8+ T cells accumulate in the tumor stroma not the tumor epithelium), and immune infiltrated (CD8+ T cells infiltrate the tumor epithelium) using a machine-learning approach in a previously published analysis [30]. Cytolytic activity was highly associated (P = 4.9 × 10− 24) with tumor-immune phenotype where activity was increased in immune excluded tumors and even more so for immune infiltrated tumors compared to immune desert tumors (Fig. 3A). EZH2 mRNA expression was significantly downregulated in immune excluded tumors compared to immune deserts and immune infiltrated tumors (P = 0.001; Fig. 3A). No clinicopathologic factors (age, stage, or residual disease) were associated with differential EZH2 expression, cytolytic activity, or tumor-immune phenotypes (Table S3, Additional file 1).
Evaluation of EZH2 expression in HGSOC TCGA samples. (a) Boxplots of expression levels for EZH2 mRNA, EZH2 protein and cytolytic activity (CYT) metric across the four molecular subtypes of HGSOC. Expression levels/scores were compared across groups with linear regression models adjusting for tumor purity. (b) Boxplots of EZH2 expression by response to therapy. Unadjusted group comparisons using Student's t-test are represented. (c) EZH2 mRNA expression in platinum sensitive and platinum resistant tumors, stratified by expression of top modifier genes CARM1 and MALAT1. Unadjusted group comparisons using Student's t-test are represented. (d) Planar filtered network of genes differentially correlated (p < 0.05, n = 1490) with EZH2 in platinum resistant compared to platinum sensitive HGSOC. Modules were identified at P < 0.05 in this network. Two distinct parent modules represent the distinct sensitive (n = 440) and resistant (n = 559) co-expression networks. Modules are identified with distinct colors for visualization. (e) Differential correlation between EZH2 and CARM1 in platinum resistant tumors. (f) GO enrichment for genes correlated with EZH2 in platinum sensitive network versus the resistant EZH2 network. mRNA and protein analyses are labeled separately. The number of genes represented in each GO Biological Process are annotated. (g) Differential correlation between EZH2 and genes that were identified in both mRNA and protein expression analysis. Statistical significance of boxplot comparisons is indicated as follows *p < 0.05, **p < 0.01, ***p < 0.0001
EZH2 expression and co-expression associated with prognosis in TCGA
We next evaluated EZH2 expression by clinical outcomes and found both EZH2 mRNA and protein levels were significantly lower in the 29% of HGSOC with platinum resistance (Fig. 3B) and were significantly associated with RTT after adjustment for tumor purity, residual disease, and molecular subtype (mRNA P = 0.003, protein P = 0.03). The association did not vary by molecular subtype for mRNA (Pinteraction = 0.44) or protein (Pinteraction = 0.46). We also examined whether the association between EZH2 expression and RTT varied by tumor immune features. The mRNA and protein associations did not vary by cytolytic activity (P interaction = 0.70 and 0.23, respectively) or by tumor-immune phenotype (P interaction = 0.85 and 0.32, respectively). Neither mRNA nor protein EZH2 expression was associated with overall survival (mRNA P = 0.44, protein P = 0.57) or HGSOC molecular subtype-specific survival.
To determine whether the relationship between EZH2 and RTT was dependent upon the expression of other genes, we tested for genes that interacted with EZH2 expression in the prognostic model. We classified 164 genes as 'modifier' genes that had a significant statistical interaction (P < 0.02; top 1%, see Table S4, Additional file 1) with EZH2 expression levels in the RTT logistic model (see Methods). Top modifier genes included CERS4 (Pinteraction = 0.001) and CARM1 (Pinteraction = 0.001). Of the modifier genes, 72% (n = 118) negatively modified or decreased the prognostic significance of EZH2, including arginine methyltransferase CARM1, and 28% positively modified (increased) the prognostic significance of EZH2 such as lncRNA MALAT1 (Fig. 3C). Given that the top modifiers CARM1 and MALAT1 affected EZH2 prognostic value in opposite directions, we investigated their co-expression and found that CARM1 and MALAT1 (also known as NEAT2) were negatively correlated (r = − 0.15, P = 0.004) across HGSOC tumors. Since CARM1 transcriptionally downregulates lncRNA NEAT1 by binding to its promoter [31], we assessed the correlation between NEAT1 and downstream MALAT1 and found a strong correlation (r = 0.74, P = 2.8 × 10− 60), suggesting CARM1 may also downregulate MALAT1. We further assessed the relationship of CARM1 with BRG1 (SMARCA4), the catalytic subunit of the SWI/SNF chromatic remodeling complex that is known to physically interact with MALAT1 and is an antagonist to the EZH2/PRC2 complex [32]. We found CARM1 was strongly correlated with SMARCA4 expression (r = 0.79, P = 4.9 × 10− 71).
We next performed genome-wide co-expression analysis for EZH2 among all HGSOC samples, and by response to therapy to identify EZH2 expression networks and how they may differ in platinum resistant versus sensitive tumors. Among all HGSOC samples, transcriptional EZH2 was positively co-expressed with 1448 genes including known cofactors (i.e. proteins that physically interact with EZH2) XRCC2, BRCA1, BRCA2, and DNA methyltransferase DNMT1 and negatively co-expressed with 990 genes including tumor suppressor gene ZBTB4 [33] (Fig. S3; see Table S5, Additional file 1). Differential co-expression analysis was performed to compare EZH2 co-expression by RTT. We identified 1049 mRNA that were differentially correlated (P < 0.05) with EZH2 in platinum resistant HGSOC compared to platinum sensitive (Fig. 3D; see Table S6, Additional file 1). This included 559 genes that were exclusively correlated with EZH2 in platinum resistant tumors (Fig. S4, Additional file 1) and 440 genes that were exclusively correlated with EZH2 in platinum sensitive tumors (Fig. S5, Additional file 1). Notably, over 80% (n = 135) of the modifier genes identified in our previous interaction analysis were also differentially correlated with EZH2 based on RTT, indicating that the change in EZH2 prognostic value is mediated by the changes in its transcriptional network. For example, the modifier CARM1 was positively correlated with EZH2 in resistant tumors (r = 0.33, P = 0.003) but not in sensitive tumors (r = − 0.05, P = 0.49), representing a distinct correlation (Fig. 3E) that coincides with EZH2 downregulation in resistance (Fig. 3C). Gene ontology (GO) enrichment analysis was performed conditionally on the hierarchical structure of the GO database to reduce redundancy and identify enriched child term biological processes. This revealed that the platinum sensitive EZH2 co-expression network was enriched for genes in immune-related biological processes (T cell activation/differentiation) and metal toxicity (detoxification/response to copper/zinc) while the network exclusive to platinum resistant tumors was enriched for transcriptional regulation processes and RNA synthesis, such as RNA polymerase II subunit POLR2F (Fig. 3F). Unconditional gene set enrichment analysis from the Molecular Signatures Database (v7.3) detected analogous biological process enrichments with larger GO parent terms detected and hence larger gene sets from co-expression networks represented (Fig. S6, Additional file 1).
We similarly performed co-expression analysis for EZH2 protein levels. Forty-nine genes had correlated protein expression with EZH2 at FDR of 15% (see Table S7, Additional file 1). Most proteins (n = 36) were positively co-expressed and included PARP2 (r = 0.46, P = 8.9 × 10− 5, FDR = 0.08). Differential correlation analysis with EZH2 identified 448 proteins exclusively correlated with EZH2 based on RTT (see Table S8, Additional file 1). Distinct from the transcriptional analysis, most differentially correlated proteins (76%, n = 339) were correlated with EZH2 in resistant HGSOC. The most significant difference in protein co-expression was observed for HDAC7 which was negatively correlated with EZH2 in resistant tumors but not correlated with EZH2 in sensitive tumors (sensitive r = 0.07, P = 0.67; resistant r = − 0.86, P = 1.4 × 10− 6). Interestingly, at the mRNA level, HDAC7 was not differentially correlated with EZH2 but was correlated with the modifier CARM1 (r = 0.14, P = 0.01). Analysis of gene ontologies found that genes exclusively correlated with EZH2 in resistant tumors were enriched for protein regulation (Fig. 3F). Strikingly, only 23 of the genes that were differentially correlated with EZH2 at the mRNA level were also differentially correlated at the protein level (Fig. 3F). Further, nineteen of these genes displayed the same direction of correlation in both mRNA and protein expression including CARM1.
EZH2 plays a critical role in the development, progression, and chemo-resistance of EOC where it has been shown to have a prognostic significance [34,35,36]. We investigated the expression of EZH2 in HGSOC immunohistochemically, and consistent with prior studies, found EZH2 expression was predictive of chemotherapy resistance and shorter survival; however, we only observed this for HGSOC with distinct morphologic features, including those without infiltrating lymphocytes (TIL-) and those with lymphovascular invasion (LVI+). EZH2 overexpression is known to correlate with biologically aggressive EOC features yet prognostic associations have been independent of these factors. Now, our analysis has revealed a mixed prognostic ability for EZH2 dependent on immunogenic and angiogenic tumor properties.
To elucidate molecular correlates underlying the observed EZH2 association with RTT, we explored transcriptomic and proteomic co-expression networks within TCGA. In contrast to findings from our TMA, high EZH2 mRNA (and correlated protein) expression was associated with platinum response and longer survival times in TCGA tumors. This finding agrees with a recent study that found EZH2 protein levels were elevated in serous tumors that responded to platinum therapy and correlated with longer disease-free survival [37]. Furthermore, the association between high EZH2 mRNA expression and better survival was replicated by microarray expression data from 616 samples compiled from Gene Expression Omnibus (GEO) datasets, including the TCGA samples evaluated in this study, and confirms the association we observed in all available public data. Notably, the authors also performed an IHC quantification of EZH2 which failed to detect an increase in EZH2 among those with better survival. Our results similarly show a discrepancy between results from different quantification methods that may be due to the lower sensitivity of IHC. The discrepancy in our results may also reflect that TCGA data were generated from bulk tumor samples which capture a wide array of tumor cells that could have heterogeneous EZH2 expression. Indeed, loss of EZH2 expression at the tumor invasion front in colorectal cancer has been associated with a more aggressive phenotype and shorter survival due to reduced proliferation and acquisition of mesenchymal characteristics [38]. Moreover, cancer stem cells exhibit reduced EZH2 expression that is associated with higher transcriptional activity and cellular plasticity [39]. Considering these findings, the lower EZH2 expression in platinum resistant HGSOC may result from more invasive cellular phenotypes and higher prevalence of stem-like tumor cells that contribute to an intrinsic resistance to chemotherapy. Conversely, the TMA was, by design, a homogeneous sampling of tumor epithelium where higher EZH2 expression may contribute to chemotherapy resistance through separate mechanisms – conceivably through immune-related pathways since TIL presence affected the prognostic significance.
Presence of TIL indicates an active host immune response to tumor antigens and has been associated with favorable prognosis [14] as well as higher rates of complete chemotherapy response [40]. In our clinical series, EZH2 was overexpressed in patients with platinum resistance and TIL- tumors, suggesting that immune-evasion underlies the association. In fact, epigenetic silencing is a critical immune-evasion mechanism enlisted by tumors. EZH2-mediated methylation has been demonstrated to repress T helper 1 (Th1) chemokines, CXCL9 and CXCL10, resulting in reduced T-cell recruitment to the tumor microenvironment [41]. Thus, EZH2 has a pivotal role in TIL- tumors where higher expression contributes to immune-evasion and consequently, as our data indicate, an increased likelihood of platinum resistance. On the other hand, TIL+ HGSOC are absent the epigenetic mechanisms involving EZH2 which may explain its diminished significance for prognosis in these tumors. Although TCGA tumors were not pathologically reviewed for TIL characteristics, we were able to incorporate a cytolytic activity score and a transcriptome-based tumor-immune phenotype to evaluate EZH2 in context of the immune features. However, we did not observe an association between EZH2 expression and prognosis that was dependent upon these immune characteristics. It is possible that the metrics we used were not able to capture the complexity of T-cell spatial and quantity features. Further, the prognostic relevancy of EZH2 expression may only be within a particular habitat. Indeed, distinct immune microenvironments coexist within the same tumor and higher variability of T-cell infiltration and type (CD8+, CD4+ and Treg cells) exists within a tumor than across tumor sites and patients [42]. Thus, it is unclear how the prognostic value of EZH2 in TIL- regions can be recapitulated with bulk samples. It is of interest to delineate the associations between EZH2, tumor immune phenotypes, and prognosis in future studies.
We observed a molecular context-dependent relationship between EZH2 expression and RTT in the TCGA data. Transcriptome analysis revealed that platinum resistant tumors exhibited lower bulk tumor EZH2 expression with low CARM1 (and high MALAT1) co-expression. CARM1 mRNA levels were also strongly correlated with the SMARCA4 (BRG1) subunit of the SWI/SNF complex, illustrating a distinct regulatory network in refractory tumors where SWI/SNF and PRC2 factors are positively co-expressed. The observed downregulation of these antagonistic complexes in association with chemoresistance is intriguing. CARM1 can methylate and displace SWI/SNF from target genes and consequently promote EZH2 placement and gene silencing [43]; however, EZH2 was prognostic in low CARM1-expressing tumors, likely precluding this as an underlying mechanism. SWI/SNF complexes both activate and repress transcription through recruitment of histone deacetylases (HDACs) and have a critical role in promoting pluripotency in somatic and embryonic stem cells [44]. Interestingly, we also observed that EZH2 protein in platinum resistant tumors was negatively correlated with HDAC7 which is transcriptionally upregulated in ovarian cancer stem cells [45]. Thus, the downregulation of both SWI/SNF and PRC2 complex factors but upregulation of HDAC7 suggests EZH2-associated platinum resistance may reflect increased tumor stemness.
Finally, our assessment of our clinical samples included a detailed evaluation of morphologic patterns at primary and metastatic anatomical sites and demonstrated that none of the architectural patterns are associated with RTT in concordance with previous reports [46]. The correlation between micropapillary pattern and LVI has been documented in other tumor subtypes [47, 48] and it was associated with advance disease stage in our analysis. Micropapillary pattern and reduced e-cadherin expression were reportedly observed more commonly in recurrent tumors and the presence of cystic component and nonpapillary architecture in non-recurrent HGSOC samples [15]. In our series, presence of cystic component nullified the possible adverse effect of papillary architecture in overall survival suggesting that cystic component might contribute to disease confinement within the ovaries and avoiding disease spread through peritoneal surfaces. Another possible favorable prognostic pattern was the presence of solid component in the absence of TIL. The so-called SET pattern (Solid-Pseudoendometrioid and Transitional) with lymphocytic infiltrates was previously described in serous tumors of BRCA mutation carriers and it is associated with better survival results [16]. Overall, tumor behavior seems to be dependent on combination of multiple factors rather than a single morphologic feature.
In conclusion, we found that the expression of EZH2 depends upon complex interactions with immunologic pathways and local tumor microenvironment that alter its prognostic interpretation and may depend upon the subpopulation of tumor cells. The association between high EZH2 expression and platinum resistance in a low TIL microenvironment may have implications for therapeutic strategies. Epigenetic reprogramming of tumors with EZH2 inhibitors has been observed to improve the efficacy of T cell therapy and PD-L1 blockade therapy [49]. Our data suggest these combination therapies might be most efficacious in TIL- HGSOC which warrants further investigation.
The tissue microarray datasets generated and analysed during the current study are not publicly available but are available from the corresponding author upon reasonable request. 'The RNA sequencing dataset analysed during the current study is available with Level 1 access from the TCGA repository (https://cancergenome.nih.gov/). The gene-level FPKM data generated in this study from TCGA Level 1 RNA sequencing reads is available from the corresponding author upon reasonable request. The proteomics dataset for the TCGA samples is publicly available through the CPTAC repository (https://cptac-data-portal.georgetown.edu/).
CPTAC:
Clinical Proteomic Tumor Analysis Consortium
EOC:
Enhancer of zesta homologue 2
HGSOC:
High-grade serous ovarian carcinoma
LVI:
PRC2:
Polycomb repressive complex 2
RTT:
Response to therapy
TCGA:
The Cancer Genome Atlas
Tumor-infiltrating lymphocyte aggregates ≥1 mm
Tissue microarray
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This work was supported in part by the National Cancer Institute (R01CA207456). The funders had no role in the conduct of this research.
Thomas A. Sellers and Ozlen Saglam contributed equally to this work.
Cancer Epidemiology, Moffitt Cancer Center, Tampa, FL, USA
Brett M. Reid, Shraddha Vyas, Peter A. Kanetsky & Jennifer B. Permuth
Biostatistics and Bioinformatics, Moffitt Cancer Center, Tampa, FL, USA
Zhihua Chen & Ann Chen
Independent researcher, Tampa, USA
Thomas A. Sellers
Department of Pathology, Moffitt Cancer Center, 12902 USF Magnolia Dr, Tampa, FL, 33612, USA
Ozlen Saglam
Brett M. Reid
Shraddha Vyas
Zhihua Chen
Ann Chen
Peter A. Kanetsky
Jennifer B. Permuth
Conceptualization and supervision, T.A.S. and O.S.; methodology, O.S. and B.M.R.; pathology review, data curation, and supervision, O.S.; clinical data curation and statistical analysis, S.V. and B.M.R.; molecular data curation, bioinformatics, and analysis, Z.C., A.C., and B.M.R.; provision of resources, J.B.P., T.A.S., and P.A.K.; writing—original draft preparation, B.M.R. and O.S.; writing—review and editing, B.M.R., J.B.P., T.A.S., P.A.K., and O.S.; All authors reviewed the finalized manuscript and approved the submission.
Correspondence to Brett M. Reid.
This study was approved by the Institutional Review Board of the University of South Florida and all patients provided written informed consent prior to enrollment for research use of data and biospecimens. All methods were carried out in accordance with relevant guidelines and regulations.
Additional file 1: Table S1:
Characteristics of Study Participants by BRCA Status. Table S2: Cross frequency table for architectural growth patterns. Table S3. Associations between Molecular and Clinicopathologic Factors in TCGA. Table S4: List of Top 1% of Modifier Genes and their Interaction Term OR and Pvalues. Table S5: Significant EZH2 mRNA Correlations from TCGA HGOSC. Table S6: Genes with significant (P < 0.05) mRNA differential correlation (r2) with EZH2 in platinum resistant (PR) versus platinum sensitive (PS) HGOSC (TCGA). Table S7: Significant EZH2 Protein Correlations from TCGA HGOSC. Table S8: Genes with significant (P < 0.05) protein differential correlation (r2) with EZH2 in platinum resistant (PR) versus platinum sensitive (PS) HGOSC (TCGA). Fig. S1. Tumor EZH2 expression by Clinical Factors. Fig. S2. Digital Hscore for EZH2 expression by tissue type. Fig. S3. Volcano plot for co-expression of 31, 237 genes with EZH2 in TCGA HGSOC (n = 336). Fig. S4. Planar filtered network of genes correlated with EZH2 exclusively in platinum resistant HGSOC. Fig. S5. Planar filtered network of genes correlated with EZH2 exclusively in platinum sensitive HGSOC. Fig. S6. Gene set enrichment analysis for co-expression networks using Molecular Signatures Database.
Reid, B.M., Vyas, S., Chen, Z. et al. Morphologic and molecular correlates of EZH2 as a predictor of platinum resistance in high-grade ovarian serous carcinoma. BMC Cancer 21, 714 (2021). https://doi.org/10.1186/s12885-021-08413-3
High-grade ovarian serous carcinoma
EZH2
Chemotherapy response