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Behind the scenes of our Rachel Roy spring campaign… the whole story here!
Me and & @twinkiebus shooting through the nebula for @rachel_roy #rrexotic - photo by @theglamourai
Smoke bombs in the desert for our Rachel Roy shoot and a funny story about how they came to be…
Finding our dreams on the open road…
Setting sun on the Salton Sea…more from our journey west here!
The great American west…
Finished location scouting for @rachel_roy ready for tomorrow's shoot! #rrexotic
In the middle of the desert, we found lemon trees!
I love a day in the desert that starts like this…
Taking pictures out the plane window… Can you guess where I'm going now?
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Nature's desert beauty… #rrexotic (at Joshua Tree National Park)
A journey west…looks like Mars!
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We have landed and hit the road! Location scouting for our @rachel_roy #RRexotic shoot next week!
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My beat in Jeff City focuses on higher education issues -- an important topic, of course, since legislators hold the pursestrings for state college. Below you'll find my resume and some references. If you need a hard-working reporter, I'll be out of Mizzou in December.
[Missouri Digital News is produced by Missouri Digital News, Inc. -- a non profit organization of journalists -- with support from the the Missouri Press Association,
and KMOX Radio in St. Louis.


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Get the Word Out - Take 2~!
This is the second round of "Get the Word Out!" - an event held back in July. Now that LaTale has launched, there is an entire new game that will need a market, wiki, guides, and maybe a home for their guild.
That's where this event comes in! Until September 31st (lol jk, 30th), put a head notice, shout, whatever the game system allows up advertising MyMMOGames.net! We're striving to reach the LaTale community, but you're free to do this in any game MMOG covers (even if it's just one of our general games forums!). You're welcome to go to other forums as well, but ensure you post in compliance with their Terms of Service! If I get complaints you'll, at the very least, be disqualified from this event.
The person with the most advertisements wins. In the event of a tie, the tie will be broken by a random drawing. The winner will have their profile featured on the front page of MMOG for the entire month of October and will win the "Event Winner" banner:
If you do this, we ask that you leave your advertisement up for a half-hour or so. Of course we won't know but hey, it's your community too! If the game doesn't allow for standing advertisements, then that's acceptable. Just post your screenshot here to be considered.
Under no circumstances are you to go to other forums, boards, or communities and spam advertisements as this will automatically disqualify you. We don't want people coming here and doing that, so please don't go other places and do that. If it's against the ToS for a particular game, please choose another.
Lol, a new challenge.
We're broke so I can't offer money - hopefully people will want the banner in their postbit =D ... or just to help us out ~_~
I'll chip in some effort, though in TO only (I dun play any other games featured in MMOG :py06:)
TO still has a sizeable player base that doesn't know where we are - lol. I log on to blast MMOG links over the megaphone before maintenances and at random points in the week =P
So i just, make an advertisement? I can do that in LaTale in TO, nothing else really.
I'll try this when i hab free time, I'll be mostly on atlantic when OB opens on the 24th.
I'll try to keep my mycamp up on Trickster with an advertisement. :3
I went into highsec on Eve and found a bunch of newbie miners and told them to love MMOG or I'd blow them to smithereens and each of them obviously feigned ignorance so I wasted them and podded thier asses for good measure
I'd love to help, but with no computer now officially and no internet, i obviously cant take part in this event.....
What if i put signs up at work (blockbuster) when we put out the UGC, nexon, OGP, Gaia, etc cards and take a picture of that... would that work? (kinda being serious here lol)
I'm not exactly sure on what we are supposed to do...
Just tell people to join MMOG?
And then what?
How would you determine a winner from that? @_@
Are we supposed to tell people to use our forum name as the referrer or something?
Turkan > hey
Turkan > why r u targetng me :(
Ethane > have you heard of MMOG
Turkan > ??
Turkan > no
Ethane > The punishment for that is death
Ethane > hope you're insured
Turkan > ur a jerk
not sure
2. Post a screenshot
3. ?????
4. Profit!
Can I kill them while I'm advertising?
A) That might be a helpful site
B) what a jerk, thats probably a virus
C) Whh, site might be nice, but hat guys a douche so screw him and his site
i used to advertise alot..before....in TO... if im training or opening a shop or not doing anything, i always put up a camp saying.. Mymmogames.net or something... or make my Shop name mymmo...etc... Incase you have seen me.. i always wear a penguin hat, i am a mage and my IGN is Shaizu
trying to do the same to LaTale.. somehow...
Join MMOGs or Die ? Q__Q
i'll put up /Microphone in Fantasia Megalo ;D
oh cool
i'll try to advertise on
more than 1 game this time
off topic
ohh new strike command?
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text:
Volkswagen Group of America Inc. is being sued in a class action consumer fraud lawsuit by a California man claiming the automakers vehicles go through motor oil at an unusually high rate.
According to Law360 (subscription required), Young Kim alleges that after he purchased a new Audi A4 in 2010, the cars low oil light came on often, even when the oil had recently been changed. He claims that service workers at the dealership told him that it was normal for the vehicle to burn through a quart of oil every 1,000 miles.
The consumer fraud lawsuit alleges that the vehicle should not burn through oil at such a high rate and that the problem should be covered under warranty, but the company refuses to do so.
Kim charges Volkswagen with breach of express warranty, fraud, unfair business practices under California law, and negligent misrepresentation.
If you or a loved one purchased a vehicle with defective components, contact Sokolove Law for a free legal consultation on consumer fraud.
Photo courtesy of Christian Giersin via Wikimedia Commons.


text:
It's all about family.
At The University of Texas-Pan American's Spring 2014 Commencement ceremonies May 10 at the McAllen Convention Center, UTPA President Robert S. Nelsen hailed the accomplishments of the 1,600-plus graduates, as well as the rest of the UT Pan American community, during the four and a half years he has been at the helm.
"I take true pride in what we as a family have accomplished in these four and a half years, especially with regard to student access and student success," he said, citing the multi-millions in scholarships, Pell Grants, and Texas Grants awarded to students. "Yes we have made access a priority and we have make education affordable. Our net tuition is the lowest in the United States. Yet we give the best darn degrees and the best darn education there is in the United States."
Nelsen went on to describe to thousands of family members and friends of the graduates who attended one of the three ceremonies a number of key grants and awards UTPA had received, new academic and student support programs that have been initiated or enhanced, and new buildings being renovated and constructed.
"But there is one important number that means most to me, that means everything to me ... we have graduated 13,904 students, yes 13,904 graduates, and we have done it as a family," he said.
Two of the graduates and members of the UTPA family received their degrees posthumously. During a solemn and silent presentation, Monette Arguelles and her son Santiago, 4, and daughter Skyler, 11, accepted the degree of Monette's husband Eddie Arguelles. Eddie, a UT Pan American IT employee who was close to earning his bachelor's degree in general studies with a major in philosophy, was struck by an alleged drunk driver in the early morning April 17 while riding his bicycle. The other degree was for Cynthia Pumarejo who died from lupus just before she was able to receive her bachelor's degree in social work. Her degree was presented to her brother Roberto Pumarejo Jr.
John Garcia, 25, who received a BA in psychology, said UT Pan American became a family for him.
Garcia was among four former foster care youth who became college graduates in the 9 a.m. ceremony. Defying dire statistics - less than 3 percent of youth who age out of foster care graduate from college - Garcia took advantage of a Texas law that waives the tuition and fees for any individual aging out of the foster care system who wants to attend a state-supported higher education institution. At UTPA, he also benefitted from the Preparation for Adult Living (PAL) Program, a partnership the University has with the Texas Department of Family and Protective Services that provides mentorship and a renewable housing scholarship to PAL students who qualify for admission and maintain an acceptable GPA.
Garcia, who entered the foster care system at age 15 and served four years in the Marines, including a tour in Afghanistan, said his PAL mentors were highly influential and always looked out for ways to help him. He called UTPA an "excellent choice" for him.
"There was this huge support system. I'm an orphan so the University became my family," he said. "It says a lot about the University to provide this level of support for this particular population that is in dire need. To have all four of us taking this huge step ....this should help other students move in the right direction."
Garcia has applied for the master of science in social work program and is pre-admitted to law school. He said he chose psychology for his undergraduate degree because foster care youth have a high incidence of PTSD (Post-Traumatic Stress Disorder).
"I have seen the needs of those in foster care and the ins and outs and ups and downs of it definitely pulled me toward that field," he said. "I want to help to improve and to give back to the community that has made it possible for us to succeed."
Rios told them three life lessons she learned: show up and excel, be positive and respectful and do something about which they are passionate.
"Your career is not going to be easy," Rios said. "There's going to be detours, there's going to be on-ramps and off-ramps, and there's going to be some failures. There is no GPS for your career."
González, who grew up in the Rio Grande Valley and helped his family pick crops while working as migrant farmers up north, said his parents told him to have a large family if he were to continue working as a migrant farmer. They also encouraged him to pursue an education. He decided to follow the latter advice.
"You're going to have people who love you, who care about you and mean well, and they want the best for you, but they're giving you advice based on their experience, and, ultimately, while they might mean well, it may be limiting for you," González said. "This is your degree, this is your life. Follow your heart, follow your passion, and be careful with some of the advice that people will give you, because it may limit your potential."
For Mayra G. Oyervides, Saturday's graduation signified another personal goal she achieved. Oyervides, who graduated with a Master of Science in biology, said this accomplishment far exceeded her expectations.
She had dropped out of high school at 16 and soon realized she had made a mistake, so she earned her general equivalency diploma, then her Associate degree in biology from South Texas College. She went on to earn her Bachelor of Science degree in biology degree from UTPA in 2009.
"It's more than I thought I would achieve, but I'm already making plans for a Ph.D. as well," Oyervides said. "Not too shabby for a high school dropout, I guess."
"Them having this faith in me that you can do it and always pushing me to strive for more drove me to have those high aspirations to do it," she said.
She also encouraged younger students to never give up.
"Education is definitely necessary, especially for us Hispanics, because we're a large minority in terms of the number of people here but, in terms of education, we're one of the lowest," she said. "I really think we need to change that because we have the capacity, we have hundreds of grads here today, most of them Hispanics, and if you had a slip up early on in your career, in high school, just don't give up on yourself. You can always do more and there's no goal that's too far-fetched."
See more of the commencement ceremonies in this photo gallery.


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I just dont have the troubles like I did with Macintosh which is why I dont miss doing work on the Macintosh when I had too but you have to realize too that people with Macintosh are the Minority too because you have more people using PC windows based computers Globally than you do Macintosh which is because of limited software choices for the Macintosh vs the PC which I know that for a fact and alot people didnt like apples operating system either.<quoted text>
Everybody is entitled to their opinion, however many of my friends are Apple users and very few ever had problems with their computer breaking down or being infected by viruses. As for myself, I never had a problem with them at all. It's the only computer I've used since I got on the internet years ago.
Now my boss is thinking about making the switch to Macintosh and was questioning me and a friend of mine (who happens to be a coworker) about Apple. All I could do is tell him of my experiences with the Mac, but my friend uses both, so he would be able to answer more of my bosses questions.
It seems all the people I know with PC's always have a problem. Every time I go to my sisters house, her computer is down because my niece plays with it and downloads all kinds of garbage; at least that's my sisters claim.
Macintosh invented most of the breakthroughs throughout the computer years. From windows to the style of the computer, Macintosh was the leader and Microsoft and others only duplicated Macintosh to fit their types of computer. Now I understand that Macintosh is doing away with the spinning hard drives and just going to complete memory. I imagine there won't be any warm-up time with those computers. Just turn it on and it's on in a second.
Who do you support for Governor in Ohio in 2010?
Posted in the Citizen Sound-Off Forum
Since: Aug 11
Location hidden
#17597 Feb 16, 2013
Since: Aug 12
#17598 Feb 16, 2013
Apples 8th or so operating system is what Bill Gates FIRST version of window s was based on you realize.......
Windows Vista was a conple steaming pile of shit. And so is windows 8.
#17599 Feb 16, 2013
Well, I guess it's how you hit it. With me, nothing but luck as far as the reliability with a Macintosh. When I bought my first computer, I did a lot of reading on it. At the time, people were standing in the cold waiting to get their hands on (I believe) Windows 95. The funny part is that all Windows 95 did was turn your PC into a Macintosh 90. But hey, whatever you're happy with.
The history of PC"s success was that Macintosh became greedy. If you wanted a Macintosh program, you had to buy it from Apple whereas Microsoft gave licenses to everybody and their mother. Because of that successful strategy, there was more availability to programs you couldn't get in an Apple computer. Eventually, Macintosh had to do the same, but by that time, Macintosh was way behind.
In spite of all that, Apple is still the leader in technology. With these I-pands and I-phones and other gadgets, they remain the leader. But then again, how much better can they make a computer than the ones we have today????
#17600 Feb 16, 2013
Actually, Apple provided better processors in the past than microsoft. Now I believe Apple uses Pentium as well. Mine has an Intel Core 2 duo, and it's plenty fast.
I highly recommend an Apple computer, but you should check out an apple store and play around with them before you make your decision. My friend and coworker who uses both says that Macintosh is better for some things but not others. So I guess it all hinges on what you plan on doing with your computer. I know that most graphic arts companies and printing companies use Macintosh.
As Indy pointed out, you can still use your Microsoft software and other programs on a Macintosh, but I don't know if that comes free. Again, if you have an Apple store near you, those people can answer any question you have about it.
#17602 Feb 16, 2013
LOL...Whoever has the biggest jock to support! Love theM BIG!! SCHWINGING!! LOL
Since: Feb 12
Location hidden
#17603 Feb 16, 2013
I played with a big old Apple at Best Buy and I have to say, they are Pretty. This one had a big White Monitor. Whole thing was around 3 grand. My Laptop has a Intel Core I5. It is nice.
Since: Feb 12
Location hidden
#17604 Feb 16, 2013
I did not like Windows Vista. I like 8 though. I have that on my Phone and 7 on the computers. Might Upgrade them to 8.
#17605 Feb 17, 2013
Looks like the topic has been changed to PC class.
Since: Aug 12
#17606 Feb 17, 2013
I bet Strickland loved Vista....
#17607 Feb 17, 2013
And that dummy Taft probably doesn't even know what it is. LOL!
It's so nice to have a real governor for a change.
#17608 Feb 17, 2013
I would still recommend and Apple Store if you have one in your area. Just don't go when something new comes out. You'll never get into the place. I found out the hard way.
#17609 Feb 17, 2013
it sure does' might as well go with the flow & see what poster complains about the topic change! lol
Since: Feb 12
Location hidden
#17610 Feb 17, 2013
You better wake up darlin', This Means You and Me.
Ohio's Governor Proposes $10,000 Tax Cut For Top 1% While ... boldprogressives.org/ohios-governor-proposes-... -...
Feb 9, 2013 ... Ohio's Republican governor John Kasich (R-OH) has proposed a new ... As you can see, the poorest 20% would have their taxes raised by $63 ...
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Feb 7, 2013 ... Kasich's tax proposal comes in wake of Romney's failed ... explains the issue facing Ohioans: "Income tax cuts will increase inequality in Ohio ...
Kasich tax proposal would further tilt tax system in favor of ... www.policymattersohio.org/tax-policy-feb2013
Feb 7, 2013 ... Kasich tax proposal would further tilt tax system in favor of Ohio's affluent ... Income tax cuts will increase inequality in Ohio and do little for our ...
Report: Kasich Tax Proposals Will Overwhelmingly Favor Wealthy ... innovationohio.org/2013/02/07/report-kasichs-... ...
Feb 7, 2013... Kasich Tax Proposals Will Overwhelmingly Favor Wealthy Ohioans ...$33,000 and $51,000 would see an increase of about $8 a year.
Since: Aug 12
#17612 Feb 17, 2013
Ohhhhh my...... since the dregs on society dont pay any tax at all and their tax wont go up I guess that translates to any break for the ACTUAL TAXPAYERS means its a break for the wealthy.
Its time for the poor to pay their fair share.!
#17613 Feb 17, 2013
Hey everyone is 2013 not 2010... I think it's time for a new topic :):):):)
Since: Aug 11
Location hidden
#17614 Feb 17, 2013
Liberal G.W. Bush didnt feel that way.
#17615 Feb 17, 2013
You mean the same GW Bush that was criticized for his tax breaks for the rich???
Since: Aug 11
Location hidden
#17616 Feb 17, 2013
yeap that one who allowed and went along with the other Modern Liberals that kept addding everything to the Federal Deficit epecially the Republicans Medicare Part D which he should have vetoed and didnt but given the other Modern Liberal Choices to pick from in 2000 & 2004 its a no brainer and we as a country would have been worst off yet I believe if one of the other 2 got elected.
#17617 Feb 18, 2013
OBAMMY to the rescue!
#17618 Feb 18, 2013
State Aid to Local Governments:
The majority of Us the Government of this Representative Republic that vote and the nonvoters that sit on their dead hands and whine; Successfully elect Obnoxious President, Vice President, Governor, Representatives of US the Government, County Commissioners, Township Trustees, Mayors, City Council, School Administrators and Board Members.
That defies USA Labor Law, The Constitution, and demands of Natural Law: what Mother Nature, God, or Whatever Power decreed to be the reality of the real world, democracy, capitalism, the US Constitution, and free, fair, and affordable commerce and common sense demands.
Demanding every Business Owner and Stockholder, corporation, farmer, business, outsourcer sweatshop, and nonprofit, tax-exempt, organization and Church; markets the cost in the wholesale and retail price of his or her product and service; of every worker, consumer, and taxpayer's wages (union contract), healthcare, pension, investment and independent business profit.
This enables every worker, consumer, and taxpayer to pay healthcare insurance premium or pay healthcare provider. Pay ALL taxes and pay for every product and service they use for life. With money derived from wages, investment profit, and independent business profit.
And enables every parent to educate, love, nurse, nurture, discipline, protect, and provide; for every child (job) they conceive. And fund schools, infrastructure, local and national security, government services, and etc.; with money derived from wages, investment profit, and independent business profit.
This makes it IMPOSSIBLE for state to aid local Governments; and makes it IMPOSSIBLE for President and Congress to avoid fiscal cliff. Makes free, fair, and affordable commerce IMPOSSIBLE; Makes funding schools IMPOSSIBLE; Makes balancing every budget IMPOSSIBLE; Makes Union workers, consumers, taxpayers, and Americas grandchildren's children life UNAFFORDABLE; and created Ohios $1.35 trillion budget deficit, the $40 trillion social security and the $16 trillion national debt. Americas grandchildrens children are responsible to pay Chinese, Foreign and Domestic Investors and Stockholders interest with this debt until they are 18 years old.
Americas grandchildrens children cannot afford life and pay this debt with the $7.25 or $9.00 per hour Government mandated labor wage in a hundred million years.
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U.S. banking regulators told the Financial Crisis Inquiry Commission that power provided by the Dodd-Frank act should help them avoid more rescues if crisis should hit major financial institutions. "The real question before us is: How did we end up with only two choices -- either bail out the banks, or watch our world sink?" said Phil Angelides, chairman of the commission.
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Zombie Typomaniac HD
By Tagstar Publishing Ltd
Open iTunes to buy and download apps.
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Spookville, Oregon is a quaint, picturesque little town with just one tiny problem… it's infested with zombies!
An entertaining take on the horror shooting genre, Zombie Typomaniac is a quick-play arcade game, which pitches a player's typing skills and quick reflexes against hordes of offbeat zombies.
In this compelling (spelling) test of nerves and skill, the player's goal is to rid the town of its zombie infestation once and for all. Help comes courtesy of a variety of bonus health and weapon drops, but when the horde is bearing down it's the player's fast fingers which bring the firepower.
Gameplay modes include a compelling story mode across 28 levels, including 5 crazed boss characters, plus a special "survival mode" for the real killer elite.
• Fast paced arcade shooter with an entertaining and simple gameplay mechanic. Lock & load! Type to shoot!
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Gov. Jerry Brown's announcement of a $248 million cut in school transportation funds has triggered a fierce response from some of the state's largest urban school districts as well as smaller rural ones. What is far less clear is how many districts will actually terminate bus routes this year.
Schools are mandated by federal law to provide transportation for special education students if called for in their individual education plans. And if students can't get to school, districts would lose state funds they would have received based on their "average daily attendance," which in turn would compound their losses.
On average, the state has traditionally paid about 40 percent of a district's transportation costs. The rest has had to come from the district itself.
The most vociferous protest came swiftly from Los Angeles Unified, which filed a lawsuit against Gov. Brown and other state officials in Los Angeles Superior Court yesterday, less than a day after Brown's announcement.
Schools were spared some of the worst "trigger cuts" many had feared. But Los Angeles Unified stands to lose $38 million if the transportation cuts go into effect, far more than any other district. The impact of the loss of those funds, the lawsuit contends, would be "catastrophic."
"We stand with our students to say enough is enough," Los Angeles Superintendent of Schools John Deasy declared. "LAUSD cannot withstand further budget cuts without adversely impacting the educational benefits offered to its students."
Among other arguments, the lawsuit contends that unlike other districts LAUSD is "constitutionally mandated" as the result of a 1981 court-ordered voluntary desegregation plan to provide transportation to 35,000 students attending district magnet schools. "For decades, the state has reimbursed LAUSD for its court-ordered desegregation expenses," the lawsuit noted.
In addition, the district is mandated by state and federal law to provide transportation to another 13,000 special education students. The district contends that:
Unless the State is enjoined from implementing the mid-year budget cuts, the District and its students will suffer irreparable harm in violation of the California Constitution.
In making the announcement of the cut, Gov. Brown urged districts to pay for transportation out of their reserves. It's likely that many districts will do so, even though in many cases their reserves are leaner than they were before the state's fiscal implosion.
In a strongly worded Dec. 12 letter, for example, an official from Tulare Joint Union High School District, with an enrollment more than 100 times smaller than LAUSD, wrote to Gov. Brown and Ana Matosantos, his finance director, explaining how the district has to transport 900 out of its 5,600 students some 1,200 miles each day across 300 square miles in its vast Central Valley district.
The letter argued that cuts in transportation funding would "force small children to walk miles to school in horrible weather," lead to some parents dropping off children at school early and leaving them unattended, or require mass layoffs of bus drivers, adding to the area's already sky-high unemployment rate.
However, just two days after sending the letter, Tulare Joint Union High's Superintendent Sarah Koligian told EdSource that her district would avoid eliminating bus routes by dipping into its reserves to cover the $300,000 it will lose from the state. But she pointed out that was an unsustainable strategy over the long term, especially coming on top of several years of relentless budget cutting in her district.
Out of the district's total transportation costs of $1.4 million, it is already paying $800,000 out of its own funds, she said. As a result of the pending cut, it will have to cover $1.1 million of the total costs this year, further "encroaching" on the district's general fund, as she put it.
Stephen Rhoads, legislative advocate for the School Transportation Coalition, a coalition of several districts, said districts have already had to make cuts in their school route offerings as a result of several years of relentless budget cutting, and that it will be hard for districts to make further reductions, especially in the middle of the school year.
He said that the cuts would fall hardest on districts serving low-income students who are dependent on school transportation, or in rural areas where students don't live anywhere near their schools. "You're taking money away from districts where parents don't have a Lexus to drive their kids to school," he said. The disproportionate impact on the poor was "unfair," he added.
And even if schools were able to preserve their bus routes, the impact on districts would still be significant. "Cuts will have to be made someplace," he said. "You either have to cut transportation, take it out of reserves or take it out of the classroom."
With its lawsuit filed, Los Angeles Unified officials yesterday declined to state whether the district would dip into its reserve to compensate for the looming loss of transportation dollars, or find some other ways to pay for them. But the implication was that it would. "The superintendent and board are going to look at all options to avoid the disruption of students during the middle of the school year," a spokesperson said.


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We report pulsed homodyne detection of squeezed pulses at a repetition rate of . Measurement of individual pulses, which were interrogated by the correlation coefficient between adjacent pulses, was realized. A homodyne detector was constructed using a low-noise, high-speed operational amplifier; it had a usable bandwidth of . We observed of squeezing in both the time and frequency domains.
© 2008 Optical Society of AmericaFull Article | PDF Article


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Profile of Mystermenace
Contact Information
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Biographical Information
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Last seen: Feb 12, 2014 - 8:33 PM
Registration Date: Apr 29, 2010
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- Omniscience and omnipotence are two separate ideas and two separate powers of a hypothetical omni-power possessing being. An omnipotent being can use...
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FRANKLIN TOWNSHIP - Many across the Garden State are still recovering after heavy rains soaked the area this week.
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Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas.
The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark® XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives.
Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 – 1.0000) and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa = 0.8993%, 95% CI = 0.8068 – 0.9919) and without the FISH equivocal cases (100%, Simple Kappa = 1.0000%, 95% CI = 1.0000 – 1.0000).
Automated BDISH applications for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.
The human epidermal growth factor receptor 2 (HER2) oncogene, located on the long arm of chromosome 17 (17q12-q21), is over-expressed or amplified in approximately 20% of breast carcinoma cases [1,2]. HER2 status in breast cancer is used as a prognostic factor, a predictive factor, and a therapy selection factor [3] for the humanized monoclonal antibody trastuzumab (Herceptin®; Genentech), which is an FDA approved drug for use as monotherapy or combined chemotherapy for treatment of breast cancer patients with amplified HER2 status. Trastuzumab adjuvant treatment for early HER2 positive breast cancer is effective for improving patient survival and cost-effectiveness analyses of such treatment have shown acceptable ratios [4-7]. However, there is a negative aspect to trastuzumab therapy, namely cardiac toxicity [3], which is possibly due to myocardial HER2 gene over-expression associated with anthracycline treatment [8] and substantial cost.
Quantitative HER2 fluorescence in situ hybridization (FISH) analyses for detecting HER2 gene amplification and semi-quantitative HER2 immunohistochemistry (IHC) analyses for detecting over-expressed HER2 protein are performed to determine the HER2 status of breast cancer patients. The optimal scoring method for determination of HER2 gene status is the use of chromosome 17 centromere (CEN 17) enumeration for calculating the HER2/CEN 17 ratio [9]. One study showed that chromosome 17 polysomy (13%) and chromosome 17 monosomy (2%) were confirmed among 147 breast cancer cases with 2+ and 3+ HER2 IHC scores [10]. Also, chromosome 17 polysomy is a key prognosis indicator for breast cancer patients. Patients with chromosome 17 polysomy and no HER2 gene amplification have better prognosis compared to patients with HER2 gene amplification [11]. Dual color FISH for HER2 and CEN 17 targets is recommended especially for borderline IHC cases [12]. However, there are additional drawbacks to conducting HER2 FISH assays beyond the requirement for a specialized fluorescence microscope and the difficulty of preserving FISH signal during a long term storage. For example, HER2 FISH testing has exhibited a higher assay failure rate in the hands of some investigators when compared to HER2 IHC testing (5% vs. 0.08%), the FISH assay procedure time is longer than the IHC assay (36 hours vs. 4 hours), and the FISH interpretation time is longer than IHC interpretation time (7 minutes vs. 45 seconds) [1]. Another disadvantage of the FISH assay is the difficulty of correlating cytomorphological aspects of the tissue sample with the gene status [13]. Furthermore, tissue slides for the dual color FISH test are still processed manually in most laboratories, which practice can introduce human errors during the lengthy assay. In fact, FISH assays may not always be performed accurately [14]. An international HER2 proficiency testing study showed that there was 20% (4 out of 20 samples) discordance with HER2 FISH testing among 5 experienced laboratories [15]. On the other hand, some reports using proficiency testing surveys conducted by the College of American Pathologists have demonstrated a much higher concordance for FISH [16].
There are alternatives to FISH for determining HER2 gene status. The chromogenic in situ hybridization (CISH) assay using a DAB chromogen and H2O2 substrate system for horseradish peroxidase (HRP)-based signal detection has been evaluated and the value of this assay for assessing HER2 status has been demonstrated [12,17-22]. CISH slides can be interpreted using an ordinary brightfield microscope without oil-immersion lenses and can provide visible tissue morphology for correlation with the HER2 gene signal. However, with the current CISH method, the assessment of the HER2/CEN 17 ratio is conducted by enumerating HER2 and CEN 17 separately using two different tissue sections.
Other brightfield microscopy in situ hybridization (ISH) methods use autometallography and enzyme metallography: 1) Nanogold® with gold enhancement in situ hybridization (GOLDFISH) [23,24] and 2) enzyme metallography or silver in situ hybridization (SISH) [25-27]. The GOLDFISH procedure utilizes the tyramide signal amplification principle and produces large clusters for amplified HER2 gene signal. On the other hand, the SISH method produces discrete metallic silver black signals. Horseradish peroxidase (HRP) of the detection system reacts with silver acetate, hydroquinone, and H2O2 and deposits metallic silver particles at the reaction site. The reaction product can be seen as discrete black dots under a brightfield microscope. Advantages of SISH include the high sensitivity for detection of single gene copies, the high resolution for quantifying DNA targets, and the high contrast with tissue counterstaining for visual separation of the signal and tissue morphology [27].
Recently, an automated HER2 SISH assay was evaluated for assessing the inter-observer interpretative reproducibility of the HER2 gene status of 99 clinical cases when compared against the reference standard FISH results [28]. Overall concordance between dual color HER2 FISH and single color HER2 SISH was 96.0% (kappa = 0.754, 95% CI = 0.518–0.993) and the discrepancies were mainly observed among tumors with the heterogeneity of tumor cell populations [28]. Advantages of the SISH assay compared to the CISH assay include that the SISH assay produces signal clusters and separately visualized discrete black dots that are easier to count in the majority of clinical samples. With SISH, the endogenous gene copies present in non-neoplastic stromal cells are also routinely and reproducibly visualized. However, like the CISH assay, the detection of CEN 17 signal cannot be performed on the same tissue section. Thus, it would be ideal to visualize both HER2 and CEN 17 on the same tissue section like two-color FISH assays. Dual ISH staining for HER2 gene and CEN 17 would be beneficial for analyzing chromosome 17 aneusomy and for delineation of cases displaying genotypic intratumoral heterogeneity.
One prerequisite for testing HER2 status reproducibly is the use of automation for conducting the test in the same manner among different laboratories located in different parts of the world. Thus, as a step toward standardizing HER2 testing, our objective was to develop an automated brightfield double in situ hybridization (BDISH) assay for simultaneous detection of HER2 and CEN 17 DNA targets on formalin-fixed, paraffin-embedded breast cancer tissue samples. Using this method, HER2 status testing can be conducted in a simplified manner for more accurately identifying the patients who are eligible for trastuzumab therapy and potentially leading to the improvement of breast cancer patient care in the future.
MCF7 and BT-474 xenograft tumors were utilized for optimizing the BDISH assay. MCF7 is a breast adenocarcinoma cell line with non-amplified HER2 status and BT-474 is a breast ductal carcinoma with amplified HER2 status (50–60 copies of HER2) and chromosome 17 polysomy [29]. Paraffin sections (4 μm) containing tissue cores of formalin-fixed, paraffin-embedded MCF7 and BT-474 xenograft tumors were placed onto Superfrost® Plus glass slides (Erie Scientific Company, Portsmouth, New Hampshire).
Ninety-four (94) breast cancer cases were used from the Cleveland Clinic Foundation and the Cleveland Clinic Lerner College of Medicine, Cleveland, OH, USA under IRB approved protocol. Tissue samples were routinely processed for paraffin-embedding after fixing with an alcoholic formalin fixative. All breast cancer cases had been previously tested for HER2 status by FISH using the PathVysion® HER-2 DNA Probe Kit (Abbott Molecular, Des Plaines, Illinois) at the Cleveland Clinic Foundation. However, it should be noted that non-consecutive tissue sections were used for FISH and BDISH analyses.
The BenchMark® XT automated slide processing system (Ventana Medical Systems, Inc., Tucson, Arizona) was used for the optimization and performance evaluation of the BDISH assay for HER2 and CEN 17 DNA targets. A protocol was established so that the entire assay procedure consisting of baking, deparaffinization, pretreatment, hybridization, stringency wash, signal detection, and counterstaining was completed as a one-step fully automated assay. Paraffin tissue sections on glass slides were baked at 65°C for 20 minutes prior to the deparaffinization step with EZ Prep™ (Ventana) at 75°C for 16 minutes. Deparaffinized tissue sections were pretreated with a combination of heat treatment with Reaction Buffer (Tris-based pH 7.6 solution, Ventana) and ISH Protease 2 or ISH Protease 3 (Ventana) to unmask DNA targets. Pretreatment conditions were chosen for optimal signal to noise ratio and tissue morphology preservation for the xenograft control slides as well as clinical case tissue slides.
Sequential ISH procedures for HER2 and CEN 17 signal detection were conducted for a complete BDISH assay (Figure (Figure1).1). Reaction Buffer was used for the washing steps during immunological detection. Liquid Coverslip™ (LCS, a hydrophobic reagent, Ventana) was used for controlling liquid evaporation throughout the assay. For HER2 gene detection, the INFORM® HER2 DNA Probe (Ventana), a dinitrophenyl (DNP)-labeled, nick-translated repeat deleted DNA probe was applied to the glass slide for co-denaturing the probe and target at 95°C. Then, the hybridization step was conducted at 52°C for 2 hours. After 3 stringency wash steps were performed at 72°C with 2× SCC (Ventana), tissue sections were incubated with monoclonal rabbit anti-DNP antibody (Ventana) for 20 minutes and then with HRP-conjugated anti-rabbit antibody for 16 minutes at 37°C. The metallic silver deposit for HER2 ISH signal was developed using silver acetate, hydroquinone, and H2O2 reaction in the presence of HRP using the ultraView™ SISH Detection Kit (Ventana). For CEN 17 detection, the INFORM Chromosome 17 Probe (Ventana), a DNP-labeled oligoprobe, was applied to the tissue sections, denatured at 95°C and hybridized at 44°C for 2 hours. Then, after 3 stringency wash steps at 59°C with 2× SSC, tissues were incubated with rabbit monoclonal anti-DNP antibody for 20 minutes and then with an alkaline phosphatase (AP)-conjugated anti-rabbit antibody for 12 minutes at 37°C. Finally, the signal for CEN 17 was visualized with a fast red and naphthol phosphate reaction using ultraView Red ISH Detection Kit. Diaminobenzidine (DAB) chromogen and H2O2 substrate reagents from the ultraView Universal DAB Detection Kit (Ventana), 5-bromo-4 chloro-3-indolyl phosphate (BCIP) substrate and nitro blue tetrazolium (NBT) oxidant reagents from the ISH iVIEW™ Blue Detection Kit (Ventana), and a ready-to-use tetramethyl benzidine (TMB) solution (Fitzgerald Industries International, Concord, Massachusetts) were also evaluated for CEN 17 signal detection of the BDISH application. Because both HER2 and CEN 17 probes were labeled with the same DNP hapten, CEN 17 signal detection was completed without DNP-labeled CEN 17 probe after HER2 signal detection to ensure that the anti-DNP antibody of CEN 17 detection didn't recognize the DNP hapten of HER2 probe.
Single or double stained tissue sections for HER2 and/or CEN 17 targets were counterstained with Hematoxylin II (Ventana) for 4 or 8 minutes and Bluing Reagent (Ventana) for 4 minutes. Counterstained slides were first rinsed with distilled water containing DAWN® (Proctor & Gamble Company, Cincinnati, Ohio) for removing LCS from slides and then rinsed with distilled water until soap was removed completely from the slide. Slides were blotted very gently with paper towels and completely dried at 45°C or 65°C in the oven for at least 15 minutes. One drop of Cytoseal™ 60 (Richard-Allen Scientific) was applied onto a dried slide and a glass coverslip was carefully placed onto the slide. Excess mounting media was removed from the slides by gently pressing the slides against paper towels. Different coverslipping methods were also evaluated for preserving the fast red staining during the assay development. BDISH results were observed with a Nikon ECLIPSE 90i microscope (Nikon Instruments Inc., Melville, New York) equipped with Nikon digital camera DXM1200F (Nikon) without oil immersion objective lenses, up to 60×. However, for presentation purposes, mainly comparing to FISH images taken with a 100× objective lens, brightfield photographs contained in this report were obtained using a 100× oil immersion objective lens.
PathVysion HER-2 DNA Probe Kit was used for the FISH test for HER2 and CEN 17 targets of xenograft tumor controls as previously described [30]. Photographs of FISH images were taken with Zeiss Axioplan 2 microscope (Carl Zeiss MicroImaging, Inc., Thornwood, New York) with Metasystems JAIM4+ CCD1 Charge Coupling Imaging Camera (MetaSystems Group Inc., Watertown, Massachusetts) at 100× using an oil-immersion lens.
Performance of the BDISH assay was compared to FISH results as the reference standard using the historical criteria for HER2 amplification with the PathVysion assay (Negative: HER2/CEN 17 < 2.0 and Positive: HER2/CEN 17 ≥ 2.0) and using the ASCO/CAP guideline criteria (Negative: HER2/CEN 17 > 1.8, Equivocal: 1.8 ≤ HER2/CEN 17 ≤ 2.2, and Positive: HER2/CEN 17 > 2.2) with or without the equivocal cases. Scoring BDISH slides was conducted by 4 observers (MK, MD, FPL, and RRT), who were experienced with scoring HER2 FISH slides. Scoring occurred at different sites and at different occasions using different microscopes. Each individual observer evaluated the set of slides at their own pace and judgement. No scores were provided by the observers when the staining quality was deemed not adequate. There was no communication among observers regarding their scoring experience of the BDISH slides. Concordance data of FISH scores vs. consensus BDISH scores among 4 observers and FISH scores vs. individual BDISH scores by 4 observers were determined using SAS 9.1 (SAS Institute Inc, Cary, North Carolina) in calculating frequency tables and Kappa statistics. The consensus among observers was defined as the agreement of three or more observers on a given observation. Scoring of BDISH assays was also analyzed for the sensitivity and specificity against FISH scores with the historical scoring method and the ASCO/CAP scoring method without the equivocal cases. Discordant cases were investigated by a non-observer (HN) for possible causes using BDISH slides.
Images of HER2 single ISH, CEN 17 single ISH, and HER2 and CEN 17 BDISH results with formalin-fixed, paraffin-embedded xenograft tumor sections are presented in Figure Figure2.2. Single copies of HER2 signal were recognized as black discrete dots in the nuclei with MCF7 xenograft tumor (Figure (Figure2A)2A) while amplified HER2 gene signals were visualized as either an increased number of HER2 signals, clusters of black dots with BT-474 tumor, and/or both (Figure (Figure2B).2B). Single CEN 17 copies were observed as red dots that were slightly larger than the black dots for HER2 genes in the nuclei with MCF7 tumor (Figure (Figure2C)2C) and BT-474 tumor (Figure (Figure2D).2D). After single staining for HER2 gene or CEN 17 was optimized, the BDISH application with sequential detection for HER2 targets followed by CEN 17 targets was tested on xenograft tumors. Single copies of HER2 genes and CEN 17 were stained in the nuclei of MCF7 tumor cells (Figure (Figure2E)2E) and amplified HER2 genes and single copies of CEN 17 were visualized in the nuclei of BT-474 tumor cells (Figure (Figure2F).2F). Because of the size difference and color contrast of black dots for HER2 gene and red dots for CEN 17, they could be visually separated even when red and black signals were co-localized in the nuclei of MCF7 tumor cells (arrowheads, Figure Figure2E).2E). When CEN 17 probe was omitted from the complete BDISH assay, there was no fast red staining on xenograft tumor sections (data not shown). Thus, the anti-DNP antibody used for CEN 17 signal detection (the second ISH detection) didn't recognize the DNP-hapten of HER2 probe signal (the first ISH detection) even though the same hapten was used for the sequential hybridization method. For image comparison of BDISH and FISH for HER2 gene and CEN 17, FISH images with MCF7 tumor and BT-474 tumor are presented in Figure Figure2G2G and Figure Figure2H,2H, respectively. HER2 genes are seen as red-orange dots and CEN 17 targets are seen as green dots.
One successful way to preserve the fast red staining was the use of a toluene-based Cytoseal 60 mounting medium placed onto completely dried tissue sections prior to coverslipping with glass coverslips. We also confirmed that the red signal was successfully preserved with the Tissue-Tek® film coverslipper method (Sakura Finetek Japan, Tokyo, Japan) after air-drying slides (data not shown). The most common method of coverslipping tissue sections stained with fast red is the use of an aqueous mounting medium. However, this method did not produce crisp fast red staining for quantitative analyses of BDISH signals (data not shown). For the second color for the BDISH application, DAB, BCIP/NBT, and TMB detection systems that produce brown, blue, and green to blue final product, respectively, were evaluated. However, they did not provide sufficient contrast against the HER2 ISH black signal (data not shown).
After optimizing the BDISH assay for HER2 gene and CEN 17 with formalin-fixed, paraffin-embedded xenograft tumor sections, we applied the assay to 94 breast carcinoma cases and scoring BDISH slides was conducted by 4 observers (MK, MD, FPL, and RRT). The consensus among observers was defined as the agreement of three or more observers on a given observation. With the historical scoring method (Negative: HER2/CEN 17 < 2 and Positive: HER2/CEN 17 ≥ 2.0) (Table (Table1),1), the consensus concordance rate was 98.9% (Simple Kappa = 0.9736, 95% CI = 0.9222 – 1.0000), the sensitivity was 96.3%, and the specificity was 100%. Individual concordance ranges were between 97.8% (Simple Kappa = 0.9466, 95% CI = 0.8736 – 1.0000) and 100% (Simple Kappa = 01.0000, 95% CI = 1.0000 – 1.0000). With the ASCO/CAP scoring method (Negative: HER2/CEN 17 > 1.8, Equivocal: 1.8 ≤ HER2/CEN 17 ≤ 2.2, and Positive: HER2/CEN 17 > 2.2) (Table (Table2),2), the consensus concordance rate was 95.7% (Simple Kappa = 0.8993%, 95% CI = 0.8068 – 0.9919). Individual concordance ranges were between 92.5% (Simple Kappa = 0.8275, 95% CI = 0.7102 – 0.9448) and 95.7% (Simple Kappa = 0.9069, 95% CI = 0.8206 – 0.9933). With the ASCO/CAP scoring method without the FISH equivocal cases (Table (Table3),3), the consensus concordance rate was 100% (Simple Kappa = 1.0000%, 95% CI = 1.0000 – 1.0000). The sensitivity was 100% and the specificity was also 100%. Individual concordance ranges were between 97.7% (Simple Kappa = 0.9442, 95% CI = 0.8678 – 1.0000) and 100% (Simple Kappa = 1.0000, 95% CI = 1.0000 – 1.0000).
Representative images of BDISH staining on clinical samples are presented in Figure Figure3.3. Cancer cells were easily identified based on the tissue morphology and assessments of HER2/CEN 17 ratios could be readily conducted. Non-amplified HER2 gene cases showed 0–4 copies of HER2 genes and 0–4 copies of CEN 17 depending on cell cycle stage and how each cell was cut within a tissue section (Figure (Figure3A),3A), while amplified HER2 gene cases showed multiple copies or clusters of HER2 genes and a few copies of CEN 17 (Figure (Figure3B).3B). Besides non-amplified and amplified HER2 cases, cases with a single copy of HER2 gene due to centromere 17 monosomy or a monoallelic deletion of HER2 gene (Figure (Figure3C)3C) and multiple copies of CEN 17 due to chromosome 17 polysomy (Figure (Figure3D)3D) were also observed.
All discordant cases, that we defined it even by one observer disagreement between the BDISH and FISH scores, were re-examined for possible causes of conflicting results and there were 9 discordant cases. All nine (9) discordant cases of the BDISH slides presented at least some degree of the genotypic heterogeneity of tumor cell populations. In general, there were two types of the tumor cell heterogeneity with HER2 gene status within the same tissue section: 1) variegated different genotype tumor cell populations in the same area of tissue section (Figure (Figure4A)4A) and and2)2) segregated tumor populations in different areas of tissue section (Figures 4B&C). Breast cancers with obvious tumor cell heterogeneity are shown as examples in Figure Figure4.4. However, the subtle genotype heterogeneity of tumor cell populations is often seen among the equivocal cases, and it also can be seen in Figures Figures3D3D &4B which show less obvious variegated tumor cell heterogeneity. Three (3) of 9 discordant cases demonstrated the segregated tumor cell heterogeneity while the other 6 cases showed various degrees of the variegated tumor cell heterogeneity.
Accurate HER2 status testing is important for identifying breast cancer patients who may benefit from receiving trastuzumab therapy. Currently, in the United States, HER2 IHC methods are most commonly used for primary screening for HER2 status, and borderline cases are subjected to dual FISH for HER2 and CEN 17 to determine the HER2/CEN 17 ratio. Because the discordance rate between local and central/reference HER2 status testing with IHC and FISH is significantly high [14,31-33], the standardization of diagnosing breast cancer cases is recognized as a very important task for improving personalized cancer patient care [3,34]. The American Society of Clinical Oncology and the College of American Pathologists has published a guideline recommendation for testing HER2 status in breast cancer [3] and the Canadian National Consensus has updated the Canadian HER2/neu testing guideline [35]. Two potential solutions for improving the standardization of HER2 status testing include: 1) automating the entire process for slide staining [36] and slide reading [36-39] and 2) consolidating the HER2 testing process within experienced laboratories and pathologists that perform large numbers of HER2 tests [15].
One way to improve the accuracy of HER2 status testing is to automate the assay procedure for HER2 IHC and HER2 FISH assays so that human errors can be diminished. HER2 IHC assays can be performed using an automated slide staining system, but HER2 FISH assays remain technically challenging and time consuming manual molecular diagnostic assays in most laboratories. An evaluator of FISH slides must have access to specialized fluorescence microscopy in a dark room. Because of unstable FISH staining characteristics, the signals of FISH slides can be bleached easily, even while reviewing and enumerating signals. Furthermore, digital images of the FISH slide need to be captured with a sensitive camera system for each patient case for the HER2 gene status record. Therefore, it is desirable to automate a tissue-based HER2 gene status test that can be observed with a regular brightfield microscope and that produces stained slides that can be archived.
While the concept of multi-color brightfield ISH applications was published in 1990's [40,41], it was a recent achievement to visualize HER2 and CEN 17 targets within the same nuclei of tissue sections with a manual dual brightfield ISH application [42]. This dual ISH application utilized TMB chromogen for HER2 gene staining. However, based on published images [28,42], TMB staining does not provide discrete signals when compared to the SISH application. The advantages of the BDISH application for HER2 gene and CEN 17 presented in the current study are: 1) the automation of the ISH application; 2) the visualization of both HER2 gene and CEN 17 targets in the nuclei of the same cell; 3) the generation of discrete HER2 gene signals; 4) the ability to reproducibly detect endogenous HER2 and CEN 17 signals in the stromal tissues and lymphocytes as a reliable internal assay control; 5) the ability to visualise signal with brightfield microscopy with non-oil immersion lenses; and 6) the capability to permanently archive the slides.
HER2 and CEN 17 probes are co-hybridized for dual color HER2 FISH. However, for the BDISH assay, because the stringency conditions for the nick-translated HER2 probe and the CEN 17 oligoprobe were different, it was necessary to conduct sequential ISH staining steps for HER2 gene and CEN 17 targets. For CEN 17 ISH, we have optimized a new detection system with an alkaline phosphatase-conjugated antibody and fast red chromogen and naphthol phosphate substrate reaction. The fast red-based detection was selected to obtain a good contrast of CEN 17 ISH signal against the discrete black dots of HER2 SISH signal. DAB, BCIP/NBT, and TMB detection systems did not provide sufficient contrast against HER2 ISH black signal (data not shown). Because fast red precipitate is soluble in organic solvents, in general, aqueous mounting medium is used for coverslipping. However, the standard coverslipping method with aqueous mounting medium on wet tissue sections did not produce tissue sections with high resolution and therefore detailed tissue structure could not be observed (data not shown). A successful method to preserve fast red staining for CEN 17 and high resolution tissue morphology was, after completely dry the slides, to apply a toluene-based tissue mounting medium (Cytoseal 60) for coverslipping with cover glass or to use a film coverslipper (Tissue-Tek® film coverslipper). Incomplete drying resulted in faint red background staining particularly around the fast red precipitate sites with this method. Interestingly, the use of aqueous mounting medium onto the dried tissue slides produced yellowish background staining on tissue sections and this method did not produce satisfactory results (data not shown).
The specificity of single ISH for HER2 gene or CEN 17 and BDISH for both targets was evaluated with xenograft tumors. HER2 and CEN 17 copy numbers have been documented previously using the FISH assay [29]. MCF-7 cells are characterized as non-amplified HER2 and chromosome 17 polysomy (3 copies of chromosome 17 per nucleus) while one of chromosome 17 with HER2 deletion (2 HER2 copies per nucleus) [29]. BT-474 cell line presents HER2 amplification with 50–60 copies of HER2 genes and 4–6 copies of CEN 17 per nucleus [29]. Amplified HER2 genes are located not only on chromosome 17, but also are translocated on other chromosomes [29]. HER2 and CEN 17 copy numbers produced with the single target ISH and BDISH methods matched with previously reported results. As both probes are labeled with the same DNP hapten, our first concern was to determine if detecting specific signal for each probe was feasible. We confirmed that the fast red chromogen detection reagents did not produce red signal when the fast red ISH was performed without the CEN 17 probe after detection of HER2 by SISH (data not shown). Thus, the SISH detection and the fast red detection can be combined to perform a sequential double ISH assay with 2 probes labeled with the same hapten. Because the sequential BDISH application uses 2 specific stringency conditions based on the length and sequences of 2 probes, it is not necessary to design 2 probes that require the same stringency for co-hybridization, like double color FISH assays.
Concordance rates between a set of gold standard dual color HER2 FISH scores and HER2 and CEN 17 BDISH scores by 4 observers were calculated for assessing the performance of the BDISH assay. There were 9 discordant cases (9.6% of the total cases) based on BDISH score disagreement with FISH scores, even by one observer. We have found that the number of equivocal cases influences the concordance rate with the ASCO/CAP scoring method. There were 4 equivocal cases based on FISH scores and all cases showed the BDISH score disagreement by at least 2 observers. A similar observation was reported with an international HER2 testing proficiency study [15]. In their study, the discordant cases (20%) were caused by the specimen having FISH HER2/CEN 17 ratios between 1.7 and 2.3 that are close to the 'equivocal' defined by ASCO/CAP HER2 scoring method (1.8 – 2.2). They also stated "equivocal cases are difficult to interpret, even highly experienced and validated laboratories" [15]. In one study, when the FISH assay was used as the primary test for HER2 status assessment of breast carcinoma cases, heterogeneity of HER2 gene status was observed in 40 of 742 cases (5%) [43]. It has been speculated that genomic and phenotypic heterogeneity of tumor cells is the main reason for the inconsistency of HER2 testing results [44]. With current study, all of our discordant cases (9/9 or 100%) displayed the tumor cell population heterogeneity: three samples showed significant segregated tumor cell population heterogeneity (Figures 4B&C) and other cases showed subtle heterogeneity of tumor cell populations that are seen among equivocal cases (4/4 or 100%). Nonetheless, since consecutive tissue sections were not used for the FISH and BDISH analyses, one can speculate that the tissue sections for the FISH and BDISH tissue sections contained tumor cell populations with different HER2 status. Further clinical evaluations of HER2 and CEN 17 BDISH application with patient treatment outcome data are required for more accurate HER2 status assessment of breast cancer patients to be obtained.
We have successfully developed an automated BDISH application for HER2 gene and CEN 17 targets in formalin-fixed, paraffin-embedded tissue sections that is highly concordant to the FISH and is reproducibly interpreted among observers. Assessment of HER2 gene status can be conducted without the use of a specialized fluorescence microscope and the time required for completing HER2 gene status assessment can be shortened significantly. Furthermore, this application has the potential to be used for other gene targets, any combination of a gene and its chromosome centromere, and tissue section-based gene assessment tests including gene translocation studies. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphology observation.
HN, BHW, ML, AEM, FG, MF, EW, TM are employed by Ventana Medical Systems, Inc. RRT and MD received grant support and honorarium for speaking from Ventana Medical Systems, Inc.
LW, ML, JP, and RRT were responsible for identifying and prequalification of the clinical cases used in this study. HN and TMG were responsible for the BDISH assay development and feasibility studies, staining the clinical samples, and preparing the manuscript draft and image data. BHW and ML were responsible for the final assay development. AEM conducted all statistical analyses for the performance of BDISH assay. FG was the study coordinator and contributed intellectual content of the study. MF designed the probes. EW, MK, MD, RRT, and TMG critiqued the assay performance with their molecular histology expertise during the assay development. FPL, MK, MD, and RRT were the observers for scoring the clinical samples. All authors contributed intellectual inputs to the study. All authors read and approved the final manuscript.
We would like to thank Vu Nguyen for instrument maintenance and Drs. Raymond B. Nagle and Guadalupe Manriquez for slide reviewing during the assay development.
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