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- SCU Home Page
- About SCU
- Admission
- Academics
- On Campus
- News & Info
- Alumni
At the Center
At Our Best: Creating a Positive School Culture
Friday, Aug. 15, 2014
Creating a school climate that is conducive to learning, safety, and community was the focus of a presentation by Dotty McCrea, principal of Mercy High School, at the Center's Ethics Camp for Catholic School Educators, held August 12-15, at Santa Clara University. The four-day workshop provided an orientation for new teachers in the diocese.
McCrea explained that the school's culture is formed by its core values and beliefs, which drive actions and influence behaviors. It's the "subtle spirit" visitors sense when they walk through the door, reflected in the way they are greeted by the office staff, the vitality of the staff, and the engagement of students.
McCrea took teachers through a series of exercises to flesh out ways to create a supportive culture in their schools and classrooms. She talked about rituals and traditions that set up a positive environment, including moments of prayer and reflection. High expectations were also cited as key to a strong school culture.
She and the participants brainstormed ways to acknowledge students, parents, and school personnel as heroes for being positive role models of kindness and respect. They also discussed ways to celebrate and to inject humor into the classroom.
McCrea urged the teachers to tap into the gifts of all their students for curiosity, imagination, sensitivity, wonder, and joy.
Posted by Miriam Schulman


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Friday 28 - Monday 31 August 2015
Throughout the year
Saturday 29 August
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Thursday 14 May
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We operate several different funds and grant schemes to help a wide range of people.
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A great range of hotels, hostels, apartments and B&B's for you.
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Thursday 10 March 2011
We recently caught up with BiPhoria a social support group for bisexual and bicurious people in Manchester.
You are renowned for being a social group for bisexual people. Can you tell us a little bit about the work you do, and the services you provide?
Our central work is providing a little bit of bi space in a world that is still mostly about being straight or gay. So we have a regular 'talky' group space, social meet-ups, and also have people who can talk about bisexuality for groups and events. We have produced a few publications and hosted day and longer bi community events too.
We started back in 1994 so we've recently turned sixteen and are the longest running bi group in the UK. A lot has changed in that time, but we still find there are a lot of people who could use a little bi space in their lives.
BiPhoria has been granted some community funding from Manchester Pride. Please can you tell us what this money will be used for and the difference it will make to your group?
The funding pays for room hire that lets our core 'talky space' group work happen. Sometimes these meetings can be quite fluffy, but sometimes a lot more serious, depending on the issues raised - it's down to what is on the minds of the people who come along. So it lets bi people and those who think they may be bi have a space to talk about problems and issues they are running into that they might otherwise not have anywhere else. A space where bisexuality is normal rather than questioned, and is just something about you rather than 'the problem'. Thank you everyone who gave at Pride last summer for helping that happen!
There's a little bit extra in the funding which helps us be visible at events like Prides around the region so people can find us.
What advice would you give to those who are in any way questioning their sexual orientation who think they might be bi?
The most important thing is: don't panic, and don't let anyone else's ideas about sexuality dictate your life. Who you fancy may have changed, but you are still the same person.
There are lots of different ways of being bi - for example, some bi people find their attractions to men and women are very similar and would say they're 'gender blind' whereas for others they get different things from different genders. Still bi!
Talk to someone you trust, or if you could use a chance to talk to people outside your regular circle of friends, come along to a BiPhoria night or drop us a line.
Can you tell us a little bit about the regular events you have an offer?
The main talky space sessions are the first Tuesday of each month at the LGF centre on Richmond Street, from 7.30pm. There's a monthly pub meet two weeks later, but the rest of our calendar is up to group members - someone posts a suggestion to our email list and before you know it we're hiking up a mountain, soaking up some cultural event, or propping up a bar somewhere.
How can other people help and support BiPhoria?
Not everyone needs a group space, but projects like ours need a high profile to reach the people who do. So you could link to our website, or get in touch for flyers and booklets about the group and bisexuality if there are places you can distribute or display them. And if you have other ideas - drop us a line on email@example.com - we'll be glad to hear from you.
And finally, what is in store for BiPhoria 2011?
It's already been a busy year, with two new publications launched - our guide to coming out and staying out, "Getting Bi In A Gay/Straight World", and a more serious one on bisexuality and mental health that comes out of some work we did with the NHS last year. We had a day-long event to celebrate sixteen years of the group and a talk about Manchester's bi history for LGBT History Month. As we head into the summer we'll be heading out of town for Prides across the North West, for Leicester's Big Bi Fun Day and for BiFest Oxford. Then it'll be a busy pair of weekends as Manchester Pride is followed a few days later by BiCon, which is kind of "Bi Pride". And a couple of weeks after that it's Bi Visibility Day on September 23rd - no idea what we'll be doing for that yet but you can be sure we will mark the date in style!
For more information:
Email BiPhoria on email@example.com
Website www.biphoria.org.uk
Twitter www.twitter.com/biphoria
Or write to: Box 101, LGF, 5 Richmond Street, Manchester M1 3HF
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subset: commoncrawl


text:
Deputy National Security Adviser for Homeland Security and Counterterrorism John Brennan.
- Filed Under
WASHINGTON - President Obama will nominate White House counter-terrorism official John Brennan to head the Central Intelligence Agency.
Brennan, a 25-year CIA veteran, would replace David Petraeus, who resigned last year after acknowledging an extramarital affair.
Obama plans to make the Brennan announcement at the same time he nominated former Nebraska Sen. Chuck Hagel to be defense secretary. ...


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The New Being by Paul Tillich
Paul Tillich is generally considered one of the century's outstanding and influential thinkers. After teaching theology and philosophy at various German universities, he came to the United States in 1933. For many years he was Professor of Philosophical Theology at Union Theological Seminary in New York City, then University Professor at Harvard University. His books include Systematic Theology; The Courage to Be; Dynamics of Faith; Love, Power and Justice; Morality and Beyond; and Theology of Culture. The New Being was published by Charles Scribner's Sons in 1955. This material was prepared for Religion Online by Ted & Winnie Brock.
Chapter 22: Love Is Stronger Than Death
We know that we have passed out of death into life, because we love the brethren. He who does not love remains in death.
In our time, as in every age, we need to see something which is stronger than death. Death has become powerful in our time, in individual human beings, in families, in nations and in mankind as a whole. Death has become powerful—that is to say that the End, the finite, and the limitations and decay of our being have become visible. For nearly a century this was concealed in Western civilization. We had become masters in our earthly household. Our control over nature, and our social planning had widened the boundaries of our being; the affirmation of life had drowned out its negation which no longer dared make itself heard, and which fled into the hidden anxiety of our hearts, becoming fainter and fainter.
We forgot that we are finite, and we forgot the abyss of nothingness surrounding us. We had gathered into our barns the fruits of thousands of years of toil. All generations of men had labored so that we, the generation of fulfillment, might tread death under our feet. It was not death in the sense of the natural end of life which we thought to have destroyed, but death as a power in and over life as the Lord and master of the soul. We kept the picture of death from our children and when here and there, in our neighborhood and in the world, mortal convulsions and the End became visible, our security was not disturbed. For us these events were merely accidental and unavoidable, but they were not enough to tear off the lid which we had fastened down over the abyss of our being.
And suddenly the lid was torn off. The picture of Death appeared, unveiled, in a thousand forms. As in the late Middle Ages the figure of Death appeared in pictures and poetry, and the Dance of Death with every living being was painted and sung, so our generation—the generation of world wars, revolutions, and mass migrations—rediscovered the reality of death. We have seen millions die in war, hundreds of thousands in revolutions, tens of thousands in persecutions and systematic purges of minorities. Multitudes as numerous as whole nations still wander over the face of the earth or perish when artificial walls put an end to their wanderings. All those who are called refugees or immigrants belong to this wandering; in them is embodied a part of these tremendous events in which Death has again grasped the reins which we believed it had relinquished forever. Such people carry in their souls, and often in their bodies, the traces of death, and they will never completely lose them. You who have never taken part yourselves in this great migration must receive these others as symbols of a death which is a component element of life. Receive them as people who, by their destiny, shall remind us of the presence of the End in every moment of life and history. Receive them as symbols of the finiteness and transitoriness of every human concern, of every human life, and of every created thing.
We have become a generation of the End and those of us who have been refugees and exiles should not forget this when we have found a new beginning here or in another land. The End is nothing external. It is not exhausted by the loss of that which we can never regain: our childhood homes, the people with whom we grew up, the country, the things, the language which formed us, the goods, both spiritual and material, which we inherited or earned, the friends who were torn away from us by sudden death. The End is more than all this; it is in us, it has become our very being. We are a generation of the End and we should know that we are. Perhaps there are some who think that what has happened to them and to the whole world should now be forgotten. Is it not more dignified, truer and stronger to say yes to that which is our destiny, to refuse to cover the signs of the End in our lives and in our souls, to let the voice of Death be heard? Amid all the new possibilities offered to us, must we not acknowledge ourselves to be that which destiny has made us? Must we not confess that we are symbols of the End? And this End is of an age which was both great and a lie. It is the End for all finitude which always becomes a lie when it forgets that it is finite and seeks to veil the picture of death.
But who can bear to look at this picture? Only he who can look at another picture behind and beyond it—the picture of Love. For love is stronger than death. Every death means parting, separation, isolation, opposition and not participation. So it is, too, with the death of nations, the end of generations, and the atrophy of souls. Our souls become poor and disintegrate insofar as we want to be alone, insofar as we bemoan our misfortunes, nurse our despair and enjoy our bitterness, and yet turn coldly away from the physical and spiritual need of others. Love overcomes separation and creates participation in which there is more than that which the individuals involved can bring to it. Love is the infinite which is given to the finite. Therefore we love in others, for we do not merely love others, but we love the Love that is in them and which is more than their or our love.
In mutual assistance what is most important is not the alleviation of need but the actualization of love. Of course, there is no love which does not want to make the other's need its own. But there is also no true help which does not spring from love and create love. Those who fight against death and disintegration through all kinds of relief agencies know this. Often very little external help is possible. And the gratitude of those who receive help is first and always gratitude for love and only afterwards gratitude for help. Love, not help, is stronger than death. But there is no love which does not become help. Where help is given without love, there new suffering grows from the help.
Help has become almost impossible in the face of the monstrous powers which we are experiencing. Death is given power over everything finite, especially in our period of history. But death is given no power over love. Love is stronger. It creates something new out of the destruction caused by death; it bears everything and overcomes everything. It is at work where the power of death is strongest, in war and persecution and homelessness and hunger and physical death itself. It is omnipresent and here and there, in the smallest and most hidden ways as in the greatest and most visible ones, it rescues life from death. It rescues each of us, for love is stronger than death.
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Publishers Weekly December 2007 Issue Features Romances
With the sniggering title "Textually Promiscuous", Publishers Weekly takes an inside look at romances and the romance community. While the title is rude, the contents are mostly positive with a focus on the diversity (not ethnic but theme) of the genre. While paranormal or otherworldly romances are popular, it appears that the historical is not down and out but never even suffered a lapse.
I was surprised to see that PW made a couple of big errors. First, it referred to JR Ward's Lover Enshrined as being released in January 2008 when the publication date has actually been moved back to June of 2008. (As an aside, this is my least favorite Ward cover). Second, in referring to online communities, it points to Romancing the Blog, but gets the web address wrong identifying it as romancingthe-blog.com instead of romancingtheblog.com. That sucks for RTB.
In any event, it is definitely worth a read.


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Arenas verdict in; Stern next
It's impractical for the league to wait until Arenas' March 26 sentencing to define the length of his suspension. Sources indicated Friday that a sanction would likely come down from Stern in the next week or so, pending the completion of a probe by the NBA security department.
Two people familiar with the situation said there were indications that Arenas already had spoken with league investigators or would soon meet with them to detail the Dec. 21 firearms incident that resulted in Friday's plea deal with the U.S. Attorney's Office for the District of Columbia. Arenas' attorney, Ken Wainstein, could not be reached for comment.
League executives and attorneys familiar with the process predict a minimum 20-game ban for Arenas, with the possibility that Stern could suspend him for the rest of the season. Arenas, suspended indefinitely by Stern on Jan. 6 for violating the league's ban on firearms on team property and for his subsequent conduct, will have missed five games by the end of the weekend. If Stern has not rendered his decision by Feb. 2, when Arenas will have missed 13 games on indefinite suspension, the sanction would qualify for an appeal to be heard by a grievance arbitrator under provisions of the collective bargaining agreement.
The Wizards, who have already expunged Arenas' image from all corners of Verizon Center, further distanced themselves from their former centerpiece with a chilling news release after Arenas' plea Friday.
"Gilbert Arenas has been a cornerstone of the Washington Wizards for six years," the team said. "We are deeply saddened and disappointed in his actions that have led to the events of this afternoon. Gilbert used extremely poor judgment and is ultimately responsible for his own actions."
Stern has the latitude under Article 35(d) of the NBA Constitution to fine Arenas up to $50,000 and suspend him for any length of time. The NBA Players Association will appeal the suspension as a matter of procedure, but sources have told CBSSports.com that any attempt by the Wizards to void Arenas' contract would be "contested vigorously."
According to a person who has read the letter Stern sent to Arenas on Jan. 6, the commissioner left open the possibility of further discipline by the team. Stern referred to his decision to indefinitely suspend Arenas as being "without regard" to any action the team might take. But proving that Arenas' actions amounted to a violation of the "moral turpitude" clause of the Uniform Player Contract will be problematic, according to attorneys familiar with the CBA and its likely interpretation by a grievance arbitrator. Aside from that, the CBA stipulates that players cannot be punished by the league and their team for the same violation, except under "egregious" circumstances.
Arenas, 28, stands to lose $8,390,441 of his $16.2 million salary if he's suspended for the rest of the season; he makes $147,200 per game, or 1/110th of his salary. In addition, there were unconfirmed reports Friday that his shoe sponsor, adidas, had decided to void his contract with them.
If the Wizards are unable to successfully void Arenas' contract or trade him – a virtual impossibility if he serves jail time or loses the rest of this season to suspension – the only other resolution would be a buyout. One rival executive predicted that Arenas, who feels scorned and isolated by Wizards' management, would be willing to accept substantially less than the $80 million he's owed over the next four seasons for the chance at a fresh start elsewhere.
Since: Oct 31, 2009
Posted on: January 17, 2010 11:09 pm
Arenas verdict in; Stern next
If your stupid enough to not register your guns and stupid enough to bring them in the locker room, then you shouldn't get any pay from anybody. Athletes get to many chances. And the ones that should get another chance never do.
Since: Dec 10, 2006
Posted on: January 17, 2010 4:21 pm
Arenas verdict in; Stern next
Jumping in on the ghetto/ bigot comments. It is unfair to brush everryone with the same brush. Some NBA players are from modest/ poor backgrounds, some are not. Some NBA players brought up in poverty are model citizens. Some bring the whole "ghetto/ everyone is against me" attitude and behaviour with them.
Once you have made your first 10 million, you can and should give it up. You can afford bodyguards if you need them. Remember your roots but once you hit the big time, there is no excuse to still live them.
Since: Jan 24, 2008
Posted on: January 17, 2010 12:36 pm
Arenas verdict in; Stern next
It's easy blowe007 to call someone a biggot, or whatever and be PC. But I challenge you to dispute anything redwings1969 said. What he said is factually true, most NBA players do come from poverty - the ghetto. And I think it's a reasonable stattement to say they "act the same way they did before making it rich
Since: Jan 24, 2008
Posted on: January 17, 2010 12:11 pm
Arenas verdict in; Stern next
Whatever the case may be, whether West had 3 guns or Gilbert had 3 guns is not the point. Why did Gilbert pretty much get sentenced already and West is playing like nothing he did is wrong. This happened in the same state, so both rules apply. I know Gilbert had the guns in the arena, but what is Stern waiting for w/ West. Let me guess, after the season so it really doesn't effect anyone.
Since: May 19, 2007
Posted on: January 17, 2010 12:02 pm
This comment has been removed.
Post Deleted by Administrator
Since: Jan 24, 2008
Posted on: January 17, 2010 11:50 am
Arenas verdict in; Stern next
"Most come from the ghetto and aside from having money still do the same things and act the same way as they did before hitting it rich."
This post above was writen by redwings1969
Wow you sir are a biggot, it is easy to see why your handle is "redwing1969" enjoy the mullet olympics of the NHL and stay out of NBA chat rooms that you have no reason to be in.
Since: Dec 10, 2006
Posted on: January 17, 2010 8:00 am
Arenas verdict in; Stern next
ooops this response to redwings.
Since: Dec 10, 2006
Posted on: January 17, 2010 7:56 am
Arenas verdict in; Stern next
I agree with everything you say. I did not predict what will happen, I am just saying what I believe would be fair. With next year and this off the books, it brings a reasonable buyout down to maybe 20- 25 million dollars. If he gets a 20 game suspension, Washington will have 80 million minus a bit as a buyout number so maybe they have to pay 50 million.
It is possible that Gilbert will quickly agree to a buyout just to get this done, but he will likely accept less if it is obvious the league and the Wizards will be taking a strong stand. If it looks like a messy fight between union and league it will motivate Gilbert to do the right thing. Part of the agreement could be that the Wizard not suspend for a year- they buy him out and everyone moves on.
Since: May 4, 2008
Posted on: January 17, 2010 1:41 am
Arenas verdict in; Stern next
Whatever the case may be, whether West had 3 guns or Gilbert had 3 guns is not the point. Why did Gilbert pretty much get sentenced already and West is playing like nothing he did is wrong. This happened in the same state, so both rules apply. I know Gilbert had the guns in the arena, but what is Stern waiting for w/ West. Let me guess, after the season so it really doesn't effect anyone.
Since: May 17, 2008
Posted on: January 17, 2010 1:35 am
Arenas verdict in; Stern next
"You have to ask yourself a few questions. How many NBA players have been suspended for half of one season and then the entire season after that? How many professional athletes have suffered the above fate? If you find examples that have, tell me how many of them didn't physically hurt anybody or themselves?"
Problem is, there is not precendent that I am aware of -- I can think of no other player that brought 4 guns into the locker room and threatend (even if jokingly) to shoot his teammate. In addition, the possession of the 4 guns were felonies in the jurisdiction he was in---he is incredibly lucky that the prosecuting attorney agreed to file only 1 charge and give hime such a good sentencing recommendation (although the judge can reject the recommendation and sentence Arenas to the max 5 years if he wants to -- unlikely but possible). That said, I don't think it will be a 2 year suspension...rest of this season is almost a certainty. The rest will likely depend on when Arenas goes to jail and gets out. I could definitely see the suspension continuing for some period of time after Arenas gets out --sort of like Michael Vick in the NFL.


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HAZARD, Ky. (WYMT) He was a familiar face on Main Street in Hazard for decades.
Joe Duncan died Friday at the Hazard ARH. He was 85.
Duncan operated Hazard Rexall Drug for many years with his wife Elizabeth.
He came to Hazard in 1952 after graduating from college and bought the drug store, which he operated until 1989.
We interviewed him about the devastating 1957 flood for a series in 2007 and at Circle T Restaurant in 2003, where he was a regular.
Funeral arrangements are expected to be made Saturday.


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The Detroit Red Wings announced today that the club has signed free agent defenseman Andy Delmore to a one-year, two-way contract. In accordance with team policy, additional terms of the deal were not disclosed.
Delmore, 32, spent the past two seasons playing in Germany for the Hamburg Freezers. He has appeared in 283 games in the National Hockey League with the Philadelphia Flyers, Nashville Predators, Buffalo Sabres and Columbus Blue Jackets. He led all NHL defensemen in 2002-03 scoring 14 power-play goals for the Predators. The LaSalle, Ontario native has registered 101 points (43-58—101) and 105 penalty minutes in his NHL career.
Delmore was the winner of the American Hockey League's Eddie Shore Award as the league's best defenseman while playing for Syracuse in 2005-06. In 335 career AHL games, he has accumulated 203 points (60-143—203) and 208 PIM.


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I have Stiff Person Syndrome and my other car is a candy apple red Rascal. Feel free to ask me about it.
Though I joked about gin, not all non-pregnant MDCers are able to drink alcohol...so, yes, cameragirl, sparkling cider it is! Kombucha is also a fine alternative...and there's a N.A. beer called Kaliber that doesn't completely suck...anybody know of a sparkling cider that isn't so darn sweet? Guess that's another thread...
I got tired of my signature, but I still love my children and husband and miss my little brother.
We have two leaders and two backups. I just have to get the forum set up, contact them about specifics of forum leading, and place guidelines. Hopefully tomorrow we'll have a live forum. :)
Thank you!
Mommy to my little Nugget
144/2014 items decluttered 2/52 projects completed
Shouldn't be much longer. Our forum leaders are discussing forum management details. :)
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Stars Photos
Crystal ball gazing: What to expect from Jim Nill's five biggest moves as Stars' GM
Photo: G.J. McCarthy/Staff Photographer
WHAT TO EXPECT FROM JIM NILL'S FIVE BIGGEST MOVES: Jim Nill has made five big moves since being named Stars general manager on April 29. So what's the best fans can expect, and what's the worst they should fear? Staff writer Mike Heika takes a look at the decisions...
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text: On Thursday 30 June 2005 09:57 pm, David O'Brien wrote: > On Thu, Jun 30, 2005 at 07:17:12PM -0400, Suleiman Souhlal wrote: > > On Jun 30, 2005, at 6:41 PM, Garance A Drosihn wrote: > > >Also, what does sysinstall run to look at these partitions? I > > >tried 'bsdlabel' to read the list of partitions, but all it would > > >say was: "a -m <architecture> option must be specified" > > > > We don't support bsd labels on ppc.. I had patches that I sent phk to > > make them work, about a year ago, but he said he'd rather not have > > them supported, because of their limits. > > At this point with FreeBSD/PPC 6.0-RELEASE happening we can't stick our > heads in the sand anymore. What type of labels do we support and which > non-Sysinstall programs does one use when adding a 2nd disk to their > system? GEOM_APPLE knows how to read the Apple labels, so I think what happens is that you have to create the partitions in OS X and then sysinstall will see them via GEOM_APPLE so that you can newfs them and stick bits on them. I believe that gpt(8) can be used on all of our archs for non-boot disks, and GPT is a much better format than bsdlabel, MBR, etc. -- John Baldwin <jhb_(_at_)_FreeBSD_(_dot_)_org> <>< http://www.FreeBSD.org/~jhb/ "Power Users Use the Power to Serve" = http://www.FreeBSD.org _______________________________________________ freebsd-ppc_(_at_)_freebsd_(_dot_)_org mailing list http://lists.freebsd.org/mailman/listinfo/freebsd-ppc To unsubscribe, send any mail to "freebsd-ppc-unsubscribe_(_at_)_freebsd_(_dot_)_org"


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>Sonora / Tuolumne News, Sports, & Weather, Angels Camp, Twain Harte, Jamestown | Union Democrat
A Jamestown man pleaded not guilty Wednesday to a charge that he killed his infant daughter, who died at a hospital in December.
Kevin Michael Boehmer, 26, was charged at a motion hearing Wednesday in Tuolumne County Superior Court with assault on a child under 8 years old resulting in death.
Boehmer was previously charged with assault on a child under 8 years old resulting in a coma or paralysis. Tuolumne County District Attorney Mike Knowles sought to amend the charge after 3-month-old Kalie Boehmer was pronounced dead by doctors at Oakland Children's Hospital on Dec. 11. The girl died two days after her father took her to Sonora Regional Medical Center because she was having difficulty breathing.
Sonora hospital staff noticed several old bruises and injuries around the child's neck and chest. They reported it to the Sheriff's Office, and deputies arrested Boehmer on suspicion of willful cruelty to a child resulting in possible injury or death.
The new charge had to be approved by a judge, because Boehmer had already pleaded not guilty to the prior charge at his arraignment.
Judge Eleanor Provost not only granted the motion but also Knowles' request for an increase in bail from $200,000 to $1 million.
The maximum sentence if convicted is life in prison with the possibility of parole after 25 years, Knowles said. Boehmer's preliminary hearing was originally scheduled for Friday but was continued until Feb. 6 at 1:15 p.m. in Department 4.
The Union Democrat is now online in a Replica E-edition form and publishes Tuesday through Saturday. E-Edition or Print Plus subscribers have full access.
If you are not a current subscriber, subscribe today for immediate access. Circulation: 209-533-3614
Weekly Arts and entertainment guide for Calaveras and Tuolumne counties
Available online every Friday or get it early in Thursday's print edition.


text:
There are currently three postulated genomic subtypes of the childhood tumour neuroblastoma (NB); Type 1, Type 2A, and Type 2B. The most aggressive forms of NB are characterized by amplification of the oncogene MYCN (MNA) and low expression of the favourable marker NTRK1. Recently, mutations or high expression of the familial predisposition gene Anaplastic Lymphoma Kinase (ALK) was associated to unfavourable biology of sporadic NB. Also, various other genes have been linked to NB pathogenesis.
The present study explores subgroup discrimination by gene expression profiling using three published microarray studies on NB (47 samples). Four distinct clusters were identified by Principal Components Analysis (PCA) in two separate data sets, which could be verified by an unsupervised hierarchical clustering in a third independent data set (101 NB samples) using a set of 74 discriminative genes. The expression signature of six NB-associated genes ALK, BIRC5, CCND1, MYCN, NTRK1, and PHOX2B, significantly discriminated the four clusters (p < 0.05, one-way ANOVA test). PCA clusters p1, p2, and p3 were found to correspond well to the postulated subtypes 1, 2A, and 2B, respectively. Remarkably, a fourth novel cluster was detected in all three independent data sets. This cluster comprised mainly 11q-deleted MNA-negative tumours with low expression of ALK, BIRC5, and PHOX2B, and was significantly associated with higher tumour stage, poor outcome and poor survival compared to the Type 1-corresponding favourable group (INSS stage 4 and/or dead of disease, p < 0.05, Fisher's exact test).
Based on expression profiling we have identified four molecular subgroups of neuroblastoma, which can be distinguished by a 6-gene signature. The fourth subgroup has not been described elsewhere, and efforts are currently made to further investigate this group's specific characteristics.
Neuroblastoma (NB) is a childhood tumour of the sympathetic nervous system, and is the most common cancer diagnosed during infancy. The prognosis of NB patients depend upon clinical factors as stage [1], age at diagnosis [2], tumour histopathology [3], and several genetic factors as MYCN amplification (MNA) status [4] and DNA index [5]. Generally, children diagnosed before the age of 18 months with a localized tumour have a favourable outcome, whereas older children with metastasised tumours show poor prognosis and low survival rate. However, MNA is proven to be strongly associated with rapid progression and poor prognosis in all patients despite age and stage of disease [4,6] and is therefore central to the risk stratification system in all clinical trial groups [7]. It is still important to emphasize that the majority of metastatic tumours do not show amplification of the MYCN oncogene, and other chromosomal aberrations are being evaluated for the International Neuroblastoma Risk Group (INRG) classification system [8].
Studies show that sporadic NB tumours can be assigned to three major subtypes based on their genomic profile, and these molecular signatures also categorize risk groups of NB patients [9]. Type 1 comprise low-risk tumours with triploid DNA content, numeric alterations, and high expression of the nerve growth factor TrkA [10,11], Type 2A involve intermediate-risk tumours with high occurrence of 11q-deletion (del11q) and 17q gain (gain17q) but no MNA, and Type 2B comprise high-risk MYCN amplified tumours with high occurrence of gain17q and 1p-deletion (del1p) [12,13]. The outcome prediction of intermediate-risk tumours still remains uncertain and some tumours cannot be definitively assigned to any of the three major groups, indicating this division to be only broadly outlined.
Genome-wide transcriptome microarray analysis enables the possibility to investigate the expression of all genes in a tumour simultaneously. De Preter and colleagues established a 132-gene classifier that discriminates the three major genomic NB subtypes reflecting inherent differences in gene expression between these subtypes [14]. Several studies have established predictive gene signatures of NB tumours [14-20], and others have focused on the differential expression of genes between tumour subsets [21-29]. The three genes MYCN [30], ALK [31], and PHOX2B [32] have been directly linked to NB pathogenesis; MYCN is amplified in a subgroup of aggressive metastasizing tumours, activating mutations of ALK or amplification is seen in approximately 7% of sporadic cases [33,31-37], and PHOX2B is mutated in a subset of familial cases and in a small percentage of sporadic cases [32,38].
In the present study, we explored subtype discoveries by unsupervised expression profiling using Principal Components Analysis (PCA). The analyses identified four distinct PCA clusters in two independent data sets, which were verified in a third larger data set by PCA and unsupervised hierarchical clustering. This study presents a new alternative way of subtype discrimination which will hopefully facilitate the search for subtype-specific therapeutic targets and the development of personalized medicine for children with neuroblastoma.
Subtype discovery by PCA
Principal Components Analysis (PCA) was performed on Affymetrix HU133A expression profiles from 17 [22] and 30 [25,39] samples, respectively. Using variance filtering, four distinct clusters appeared (p1-p4, Figure 1). Among the 414 (De Preter data set) and 716 (McArdle/Wilzén data set) genes which defined the clusters in the two test data sets, 226 genes overlapped between the two gene lists (Additional file 1). By cross-validation using a "leaving-one-out" strategy we found our cluster assignments to be relatively stable with only a few exceptions. The De Preter data set showed instability of three samples which shifted cluster belonging in up to 30% of cross-validations. The McArdle/Wilzén data set showed instability of two samples which shifted cluster belonging in one out of 30 cross-validations, respectively. In order to check the robustness of the clustering we made use of the cortex (n = 3) and neuroblast samples (n = 3) from the De Preter data set which were investigated by PCA in relation to the 17 NB samples. As expected, the three cortex samples formed a distinct cluster separated from the neuroblasts and NB tumour samples (Additional file 2).
Figure 1. PCA plots of two parallel cluster analyses. Principal Components Analysis (PCA) of the two test groups using a variance cut-off of 0.4. A. HU133A De Preter data set (414 variables, 17 tumour samples, [22]). B. HU133A McArdle/Wilzén data sets (716 variables, 30 tumour samples, [25,59]). Upper panel: PCA clusters, samples (spheres) are joined by nearest Euclidean neighbour. Lower panel: Expression of six NB associated genes Red = High expression; Green = Low expression. The colour scale is based on standard deviations (SD) and ranges from +2 SD (red) to -2 SD (green).
Additional file 1. PCA loadings from the De Preter and McArdle/Wilzén data set. Column 1-6: Variables (genes/probe-sets) and their PCA loadings for Principal components 1, 2, and 3 (PC1, PC2, PC3) in data-set 1 and 2 (De Preter and McArdle/Wilzén respectively). Common variables: Genes/probe-sets that were present in the PCA analysis of both data-sets.
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Additional file 2. PCA of cortex, neuroblast and NB samples. Unfiltered Principal Components Analysis (PCA) of the De Preter data set (7438 variables, 23 samples. Colour codes of spheres: Red = neuroblastoma tumour specimens; Blue = Neuroblasts; Green = Cortex.
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By Fisher's exact test, MNA and del1p were found to be significantly more frequent in PCA cluster p3 (p = 0.018 and p = 3.9E-04, Fisher's exact test, table 1, Figure 2A). High stage (INSS stage 3-4), poor outcome, and gain17q were observed in higher frequencies in PCA clusters p2, p3 and p4 (Figure 2A) compared to cluster p1. The frequency of del11q was considerably higher in PCA clusters p2 (data set 2 p = 0,012, Fisher's exact test, table 1) and p4 (p = 0.05, Fisher's exact test, data set 1, table 1). Nineteen out of 47 tumours from both data sets showed del11q, and among those 14 were found in PCA clusters p2 and p4 (Figure 2A).
Table 1. Characteristics of the four PCA clusters(p1-p4)
Figure 2. Frequency of prognostic factors and survival probability in PCA clusters. A. The frequency of prognostic factors in the four PCA clusters p1-p4 for the De Preter data set (n = 17) and McArdle/Wilzén (n = 30). High stage (3-4) = INSS stage 3 and 4; DOD = Dead of disease; MNA = MYCN amplification; Del = deletion. Significant occurrence of prognostic factors in one PCA cluster compared to the others is marked with stars (Fisher's exact test). n = the number of samples in each subgroup. B. Kaplan Meier survival curves of PCA clusters (p1-p4) from three data sets (De Preter, McArdle, and Wilzén). OS = Overall survival (n = 43). EFS = Event-free survival (n = 35). Chi-square significance by Log-rank (Mantel-Cox).
With the intention to explore the expression of genes that have previously been associated with NB, we performed a mining of gene lists from literature. Starting with 15 gene lists [14-16,18-29], 212 genes were found to be present in at least two of the lists, and among those 157 were present in all three data sets (Additional file 3). A PubMed search was performed on the Gene Symbol in co-occurrence with the terms "neuroblastoma" and "gene expression" (Additional file 4) resulting in 30 genes with hits in PubMed. Out of these 30 genes, six NB-associated genes were selected; i) the pre-disposition genes ALK and PHOX2B, ii) MYCN and CCND1which are amplified in 20-35% and 3-6% of sporadic neuroblastomas respectively [13,30,40], and iii) NTRK1 and BIRC5 which have been found to be differentially expressed between subsets of NB [41,42]. All six NB-associated genes were found to be differentially expressed between PCA clusters (Figure 1), which was statistically confirmed by a one-way ANOVA (table 1) and a multiple comparison post-hoc test (Additional file 5). ALK and BIRC5 were found to be up-regulated in cluster p3 (fold > 2.5) and these genes also showed elevated expression in cluster p2. This was in contrast to clusters p1 and p4 in which ALK and BIRC5 were found to be down-regulated (table 1, table 2). Also, a 5 times up-regulation of MYCN was found in cluster p3 in comparison to clusters p1 or p4. The opposite effect was found for NTRK1 which was highly expressed in cluster p1 (10-fold, table 1) and specifically down-regulated in cluster p3 (16-fold, table 1). Cluster p4 consistently showed low expression of all six NB-associated genes compared to the other clusters (table 1, table 2).
Additional file 3. Workflow of the study. Step 1: Subtype discovery by unsupervised PCA of two data sets (De Preter and McArdle/Wilzén) from three microarray expression studies (upper panel). Data-mining of gene lists from literature, resulting in the selection of 6 NB-associated genes (lower panel). Step 2: Defining the 74-gene subtype discrimination gene set by SAM (upper panel). Verification of subgroup existence by hierarchical clustering and PCA in a third data set (Wang) using the 74-gene set (lower panel).
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Additional file 4. Gene lists from literature & hits in PubMed. A. List of 15 expression studies used for the data-mining. B. PubMed searches of 157 and 30 genes respectively. PubMed searches were performed as follows: Search 1(left): 157 genes, search term "Gene Symbol"[TIAB] AND "Gene expression"[MeSH Terms] AND "neuroblastoma"[MeSH Terms]. Search 2 (right):30 genes, search term "Gene Symbol"[TIAB] OR "Alias name"[TIAB]) AND "Gene expression"[MeSH Terms] AND "neuroblastoma"[MeSH Terms]. The six NB-associated genes ALK, BIRC5, CCND1, MYCN, NTRK1, and PHOX2B were selected for further analysis (see text for details).
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Additional file 5. Multiple comparisons by Post hoc test (Tukey). Gene expression of ALK, BIRC5, CCND1, MYCN, NTRK1, and PHOX2B in PCA clusters p1-p4 of the two data sets De Preter (left) and McArdle/Wilzén (right) was analysed by a Post-hoc test (Tukey). Significance level is marked by a grey colour scale.
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Table 2. Characteristics of cluster p4
According to Kaplan-Meier, overall survival (OS) and event free survival (EFS) rates were significantly different between the four clusters (OS p = 2.24E-04, EFS p = 0.019, Log-rank, Mantel-cox). The lowest survival probabilities were found in PCA clusters p3 and p4 with a 5-year OS rate of 50% and 62.5% respectively, and an EFS rate of 22.2% and 25% at 5 year from diagnosis (Figure 2B). In contrast, none out of 14 patients with tumours belonging to PCA cluster p1 died from disease, and the survival probability in this cluster was 100% at 5 years from diagnosis.
Verification by hierarchical clustering and PCA
In order to verify the existence of the four groups, a discriminative gene set was defined and applied to a third independent data set. First, the p-clusters in data sets 1 and 2 were integrated by reassignment of tumours based on their 6-gene expression profile (r1-r4, Additional file 6). Rules for the r-group assignments were defined by standard deviations (SD) of expression levels and applied to both data sets. The r1-r4 representative of the p-group assignments was found to be very stable (table 3). However, three cases from the De Preter data set and seven cases from the McArdle/Wilzén data set could not be assigned to any r-group based on the rules, resulting in two data sets of 14 and 23 tumour samples, respectively. Next, four Significance Analysis of Microarray (SAM) tests were performed by multiple comparisons on the two data sets separately. The four SAM output gene lists from the two test data sets were compared to generate combined lists of overlapping genes. From the overlapping lists, 30 genes with the highest combined fold change for each of the four contrasts were selected, generating a set of 98 genes. Out of the 98 genes, 74 were present in the third data set (Wang). Third, unsupervised hierarchical clustering and PCA was performed on the Wang data set using the 74 discriminative gene set.
Additional file 6. Rules and assignments of r-groups. Rules for r-group assignments (upper table): Groups (r1-r4) were defined based on the standard deviation (sd) of expression for the six NB-associated genes. R-Assignments of samples from data set 1 and 2 into r-groups (lower table): Expression sd intervals of 5 out of 6 genes had to be in agreement with the rules for each r-group in order to be categorized.
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Table 3. r-groups vs. p-groups
The unsupervised hierarchical clustering of the 101 NB samples clearly divided tumour cases into four distinct subgroups (Figure 3A). Ninety samples were clearly allocated into one of the four hierarchical clusters (h1-h4) based on the dendogram (Figure 3A), and the remaining 11 samples were assigned to a cluster based on the nearest Euclidian neighbour in the PCA (Figure 3B). All the 17 samples assigned to the hierarchical cluster 3 (h3) were stage 4, MYCN amplified tumours with high frequency of del1p (Figure 3A, table 4). Samples assigned to hierarchical clusters h2 and h4 consisted of high stage tumours (stage 3-4) with high frequency of del11q without MYCN amplification, thus corresponding to the genomic subtype 2A (table 4). Tumours of clusters h2, h3, and h4 all show high content of gain17q.
Figure 3. Hierarchical clustering using a 74 discriminative gene set. A. Unsupervised hierarchal clustering of the Affymetrix HGU95Av2 Wang data set (102 tumour samples in total [28]) by the 74-gene set. The heat map colour scale is based on standard deviations (sd) and ranges from +2 sd (red) to -2 sd (green). The five gene clusters seen are named g1-g5. Status of eight prognostic factors is shown by black and white squares in the lower part: Outcome = Dead of disease; MNA = MYCN amplification; Del = deletion. Black = event, White = No event, Light grey = Not determined. INSS stage is marked as follows: Black = stage 4, dark grey = stage 3. The four hierarchical subgroups seen in the heat map are marked by colour dots: Green = Hierarchical cluster 1 (h1), Orange = h2, Blue = h4, Red = h3, Grey = Human fetal brain. B. PCA plot of the 74-classifier gene-set. Left panel: the four hierarchical clusters colour-coded according to the Hierarchical clustering (see above). Right panel: three INSS stages: Green = stage 1, Blue = stage 3, Red = stage 4, Grey = Human fetal brain. C. Kaplan Meier survival curves of hierarchical clusters (h1-h4) from the Wang data set. OS = Overall survival (n = 92). EFS = Event-free survival (n = 92). Chi-square significance by Log-rank (Mantel-Cox).
Table 4. Expression subgroups vs. Genomics subgroups
Patients of cluster h3 showed the worst outcome, with 10 out of 16 dead of disease (2 were lost for follow up) and a survival probability of 36.5% at 5 years from diagnosis (Figure 3C). Patients of cluster h2 also showed a worse outcome, with 10 out of 25 dead of disease and an OS rate of 58.4% at 5 years from diagnosis. In the fourth cluster h4, 3 out of 14 patients died from the disease (6 patients were lost for follow-up), and showed an OS probability of 50.5% at 5 years from diagnosis. The lowest death and relapse rates were seen in hierarchical cluster h1, which showed a 91.1% OS rate and an 88.9% EFS rate at 5 years from diagnosis (Figure 3C). A PCA of the 74 discriminative gene set in the 101 samples shows that the hierarchical subdivision overlaps well with INSS stage subdivision, and that the human fetal brain sample clusters to the hierarchical group h4 (Figure 3B).
Five specific gene clusters (g1-g5) showing differential expression in the four h-groups were noted (Figure 3A). The eighteen genes in cluster g1 mainly involved nervous system maintenance and developmental genes (e.g. NTRK1, DBH), and those were highly expressed in the favorable h1 group. The h2 and h3 groups showed high expression of a gene cluster encoding cell cycle related proteins (g2). Gene cluster 3 (g3) comprised MYCN as well as nine MYCN/c-MYC downstream targets and was highly expressed in the MNA-specific sample group h3. The fourth gene cluster (g4) was specifically over-expressed in tumours of the h4 group and comprised 22 genes which were all found to be involved in nervous system development (e.g. ERBB3, GAS7, GPC3, SOX10), nervous system maintenance (e.g. ATP1A2, COL9A3, FXYD1), or associated to CNS in other ways (e.g. ASPA, CAPN3, MT2A, SERPINA3, SGPL1). The fifth gene set (g5) which was highly expressed in sample group h3, and elevated in sample groups h2 and h4 (Figure 3A) comprised five genes (ARHGEF10, CUX2, DUSP4, LMO3, and PHGDH) with different functions.
Validation of PCA clusters and the 6-gene signature
A PCA of unfiltered global transcripts of the three data sets clearly confirmed the existence of four distinct subgroups (Additional file 7). The PCA loadings from data set 2 (McArdle/Wilzén) were utilized to plot the other two data sets (De Preter and Wang). Next, a back-check test was performed by using the PCA loadings from data set 1 (De Preter) to plot the other two data sets (McArdle/Wilzén and Wang).
Additional file 7. PCA validation of p- and h-groups using unfiltered expression data. Principal Components Analysis (PCA) of unfiltered global expression data (4728 genes) from three data sets (De Preter, McArdle/Wilzén, and Wang). A. PCA plotted by loadings generated from the McArdle/Wilzén data set. B. PCA plotted by loadings generated from the De Preter data set. Cases (spheres) are coloured by their group assignments: Green = p1/h1, Orange = p2/h2, Red = p3/h3, Blue = p4/h4.
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Also, the 6-gene signature (ALK, BIRC5, CCND1, MYCN, NTRK1, and PHOX2B) was verified to be sufficient for subtype discrimination of both the PCA clusters (p1-p4) identified in data sets 1 and 2 (De Preter and McArdle/Wilzén) and the hierarchical clusters (h1-h4) identified in the Wang data set (Figure 3). Moreover, the 6-gene expression pattern seen in the four hierarchical clusters corresponded well with the expression pattern found in the four initial PCA clusters (Figure 1 and Figure 4B). In addition, the 6-gene set specifically separated the human fetal brain specimen from the other NB tumour samples (Figure 4B), which was in contrast to what could be when seen using the 74-gene set (Figure 3A). A comparison between the expression-based p- and h- group assignments and the genomic-based subtypes showed that the p1 cluster corresponded well to the favourable subtype 1, the p2 to the 2A type, and the p3 to the MNA-specific subtype 2B (table 4). Most tumour cases of the p4 cluster were found among the genomic subtype 2A, although a few p4 cases were also found among other genomic subtypes (table 4). Approximately the same pattern could be seen when comparing the expression-based h1-h3 assignments of data set 3 (Wang) to the genomic subtypes (table 4). However, in the Wang data set a considerable higher number of cases assigned to the fourth cluster h4 were found among the favourable genomic subgroup Type 1 (table 4).
Figure 4. Verification of 6-gene signature. A. PCA of the six NB associated genes ALK, BIRC5, CCND1, MYCN, NTRK1, and PHOX2B on three data sets: De Preter (left panel), McArdle/Wilzén (middle panel), and Wang (right panel). Samples (spheres) in the two test data sets (De Preter and McArdle/Wilzén) are coloured by the PCA clusters: Green = p1, Orange = p2, Red = p3, and Blue = p4. Samples (spheres) in the Wang data set are coloured by the hierarchical subgroups: Green = h1, Orange = h2, Blue = h4, and Red = h3, Grey = Human fetal brain. Human fetal brain (marked with an arrow) is distinct from the 101 NB samples. B. Expression heat map of the 6-gene signature of the Wang data set. The colour scale is based on standard deviations (SD) and ranges from +2 SD (red) to -2 SD (green). Samples are presented in the same order as in Figure 3A.
In order to check the previously reported relationship of the MYCN and c-MYC downstream effects we investigated the expression patterns of the MYC-target genes in the four identified groups (p- and h-groups, Additional file 8). Pearson correlation tests showed MYCN and c-MYC to be significantly negatively correlated in all three data sets (table 5). MYCN was found to be specifically over-expressed in the MNA-specific group p3/h3. In contrast, c-MYC over-expression could be seen in a subset of tumours of the p1/h1, p2/h2, and p4/h4 subgroups, but not in any of the tumours assigned to the MNA-specific group p3/h3. The expression pattern of the MYCN/c-MYC downstream targets AHCY, DKC1, FBL, GMPS, and PAICS were found to preferentially follow the MYCN expression levels, and thus most highly expressed in the p3/h3group. Also, a slightly elevated expression of the MYCN/c-MYC downstream targets could be seen in group p2/h2 (Additional file 8).
Additional file 8. Expression heat map of MYCN, c-MYC and MYCN/c-MYC downstream targets. The two test data sets De Preter (n = 17, Upper left panel) and McArdle/Wilzén (n = 30, lower left panel) are divided into four PCA clusters (p1-p4), and the verification data set Wang (n = 102, right panel) is divided into four hierarchical clusters (h1-h4). The heat-map colour scale is based on standard deviations (sd) and ranges from +2 sd (red) to -2 sd (green). Status of prognostic factors is shown by black and white squares to the right of each panel. Stage/DOD: Black = INSS stage 4 or dead of disease, Dark grey = INSS stage 3, White = Low INSS stage (stage 1 or 2) and alive, Light grey = Not determined.
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Table 5. Correlations MYCN and c-MYC
A large number of publications prove that cancer can be classified through gene expression profiling. Principal components analysis (PCA) is a useful tool to reduce the dimensions of data to be able to identify and visualize hidden patterns. PCA has been widely used in genome expression studies to discriminate tumour subtypes. For example, Yeoh and colleagues identified prognostically important subtypes and a novel subgroup of pediatric acute lymphoblastic leukaemia (ALL) by PCA of gene expression data [43]. In order to develop more effective and less toxic cancer treatment it is necessary to identify and correctly classify the molecular subtypes, as well as to unravel the underlying oncogenic driving pathways for each type.
In the current study, subtypes of neuroblastoma were explored by expression profiles from four microarray studies [22,25,28,39]. In the first step, PCA was performed on two independent data sets and four distinct clusters were identified in both sets. Prognostic factors such as high INSS stage, MNA, and del1p, differed significantly between clusters. Three of the four clusters (i.e. p1-p3) corresponded well to the previously established genomic subtypes 1, 2A, and 2B. Remarkably, a fourth novel cluster (p4) with a considerable different expression profile appeared independently in both data sets, and has not been described elsewhere. This new cluster was found to encompass mainly high stage tumours with poor outcome and high frequency of del11q and del1p, but low frequency of MNA. In data set 1 (De Preter) all 3 cases assigned to cluster p4 were found to be of clinical INSS stage 4, and in data set 2 (McArdle/Wilzén) 3 out of 5 patients died from disease. However, samples assigned to the p4 cluster showed a significantly lower expression of MYCN and ALK compared to the MNA-specific cluster p3 (table 2), which indicate an alternative progression pathway within tumours assigned to the fourth cluster.
In the second step, the existence of four groups could be verified by an unsupervised hierarchical clustering and PCA of a third data set (Wang data set [28]) using a discriminative gene set of 74 genes from the first step. All tumour samples assigned to the h3 cluster were MNA tumours of clinical stage 4, and tumours assigned to the h2 and h4 clusters comprised high stage tumours with no MNA but with high content of del11q tumours. The lowest survival probability and highest relapse rates were seen in cluster h3, followed by clusters h2 and h4 in descending order (Figure 3C). A comparison between the survival probabilities of the hierarchical and PCA sub-divisions indicated that the h4 cluster constituted less unfavourable tumours compared to those assigned to the p4 cluster. The heterogeneous results seen between data sets may be explained by the small number of cases in data sets 1 and 2 as well as different tumour cohorts. As for example, the Wang data set does not include any clinical stage 2 tumours which might indicate that the cases in this data set were not randomly selected. This would of course affect the subgroup characteristics and might explain the divergent outcome seen in patient cohorts from the different datasets. Also, since the fourth subgroup (p4, h4) is a very small group it is difficult to say if the frequencies of different genetic alterations are stably distributed between the subgroups. Moreover, six patients of the h4 cluster in the Wang data set were lost for follow up, which makes it difficult to draw any major conclusions.
The hierarchical clustering also identified five gene clusters. Nervous system developmental genes, including NTRK1 and DBH were found to be highly expressed in the favourable tumour cluster h1. Cell-cycle related genes including BIRC5, CCNB1 and MCM-genes were found to be highly expressed in clusters h2 and h3. Not surprisingly, the MYC gene cluster (g3) was specifically found to be over-expressed in the MNA-specific group h3. Westermann and colleagues recently defined a core set of MYCN/c-MYC downstream target genes which were associated with malignant progression in NB [44]. In line with their results, we found MYCN and c-MYC to be significantly negatively correlated in all three data sets. We also wondered whether the c-MYC over-expression could be specifically connected to any of the four groups. However a heat map showed the c-MYC over-expression to be evenly distributed among all groups except for the MNA-specific group p3/h3 (Additional file 8). The transcription factor LMO3 (LIM domain only 3) found in gene cluster g5 has been significantly associated with a poor prognosis in NB [45]. This is concordance with the present study in which the highest expression of LMO3 was found in the most unfavourable tumour group h3. Interestingly, LMO3 has been shown to interact and act as a co-repressor of p53 [46].
The fourth novel tumour group (h4) was found to be characterized by high expression of several brain-specific and nervous system developmental genes. The Erbb receptors (e.g. Erbb3) and the SoxE family (Sox8, Sox9 and Sox10) are essential for development of the sympathetic nervous system and the development of neural crest cells. Interestingly, Leon and colleagues reported that Sox10 and Phox2b act together with the NK2 homeobox Nkx2-1 to modify RET signalling and suggest this interaction to contribute to HSCR (Hirschprungs disease) susceptibility [47]. The growth arrest-specific 7, Gas7, is regulated by Sox9 and the ERK1/2 MAP kinase and is involved in chondrogenesis, and has been reported to form a MLL/GAS7 fusion protein in a pediatric case of B-cell acute lymphoblastic leukaemia [48]. In addition, the g4 cluster comprised the suggested 3p tumour suppressor gene SEMA3B [49]. The subgroup discrimination properties of SEMA3B found in the present study, as well as the significantly lower expression observed in tumour groups p2/h2 and p3/h3, could support its tumour suppressor function.
The validation test of the four PCA clusters using unsupervised and unfiltered global transcripts clearly shows that the four subgroups exist in all three data sets (Additional file 7). Moreover, the PCA of the 6-gene signature in all three data sets convincingly show that this expression profile is sufficient for NB subtype discrimination (Figure 4A). Overall, our results indicate that the most unfavourable group displaying MNA (corresponding to Type 2B) and the most favourable group with high NTRK1 expression (corresponding to Type 1) can be easily discriminated by their expression profiles. Moreover, our data indicates that the del11q tumours (corresponding to Type 2A) are divided into at least two expression subgroups (see table 4). Interestingly, the existence of two del11q expression subgroups has recently been reported by two other research groups [23,50]. Fischer and colleagues studied the gene expression patterns of del11q tumours divided into two clinical groups of favourable and unfavourable biology using their previously described prognostic 144-gene expression classifier. They found that the clinical groups clustered using unsupervised PCA and hierarchical clustering [23]. Also, Buckley and colleagues identified a 15-miRNA signature that discriminates two distinct biological subtypes of del11q tumours [50]. Our current study differs from these two studies in one important aspect- it does not rely on any prior subtype division (e.g. genomic subtypes, clinical groups etc.), which means that it is entirely unbiased. As stated by Fischer and colleagues the 11q-deletion is most likely a secondary event, and it is possible that the decision between favourable and unfavourable neuroblastoma is made by a yet undefined transformation event, for example ALK. This hypothesis is completely consistent with our finding, where ALK expression is significantly elevated in subgroup p2/h2 (see Figure 1 and 4B). In order to clarify and relate our subgroup discoveries to these recent findings, we performed a PCA of the Wang data showing tumours marked by their del11q status and coloured by their h-group belongings (Figure 5). Deletion of 11q was found to be distributed through all tumour groups except for the MNA-specific h3-group in which only two cases with 11q-deletion could be found (Figure 5A). In line with Fischer et al. [23] we filtered out all cases with MNA and/or del1p, and ended up with a PCA on 74 cases (Figure 5B). This left us with three expression groups of del11q tumours, one favourable group (h1), and two unfavourable groups (h2 and h4, see Figure 3C). In the last step we removed the h4 expression subgroup, leaving us with two groups of del11q tumours, one favourable (h1) and one unfavourable (h2). These results suggest that our subgroup discoveries are not contradictory to the findings by Fischer et al. [23] and Buckley et al. [50], but rather indicate that there are three del11q expression subgroups instead of two; one favourable with high NTRK1 expression (h1), one unfavourable with high ALK, BIRC5 and CCND1 expression (h2), and one smaller group (h4) characterized by high expression of nervous system developmental genes (e.g. ERBB3, SOX10).
Figure 5. Del11q groups of the Wang data set. Unfiltered PCA plots of the Wang data set (101 NB samples, 7542 genes). The four hierarchical clusters (h-groups) in panels A-C (left) are colour-coded as follows: Green = h1, Orange = h2, Red = h3, Blue = h4. Del11q genetic aberrations in panels A-C (right) are colour-coded as follows: Black = 11q-deletion, off-white = No 11q-deletion, White = Undetermined. A. PCA of all 101 neuroblastoma cases. B. PCA of 74 cases without MNA and Del1p C. PCA of 55 cases of the hierarchical groups h1 and h2 without MNA and Del1p.
The discriminative power of the six NB genes strengthen the fact that these genes are indeed important in neuroblastoma development. ALK was recently recognized as the NB predisposition gene and has thereafter also been found to be affected in sporadic tumours, either though mutations of the tyrosine kinase domain or by genomic amplification [31,34-37]. Interestingly, Passoni and colleagues investigated the ALK expression and protein phosphorylation status and found that over-expression of either mutated or wild-type ALK defines poor prognosis patients [51]. In this study, we found elevated expression of ALK in the p2/h2 group, and the highest expression level was found in the MNA-specific group p3/h3. Moreover, various cell-cycle related genes, including BIRC5 and CCND1, were found to be highly expressed in sample groups p2/h2 and p3/h3, but low in group p4/h4. The CCND1 gene region on 11q has been shown to be amplified in a subset of primary neuroblastic tumours [13,40], and several cases have been found to show an extensive over-expression of cyclin D1 which correlates with histological subgroups [52]. Moreover, CCND1 is used as a marker for minimal residual disease of NB [53]. The anti-apoptotic gene BIRC5 (also known as survivin) is located in the often gained region on 17q (gain17q), and has previously been found to be associated with poor prognostic factors and low survival probability in NB [41,54]. Recently, Eckerle and colleagues found that BIRC5 is a direct transcriptional target of activating E2Fs, and that BIRC5 is indirectly induced by N-myc [55]. These findings are supported by the significantly higher expression of both BIRC5 and MYCN found in tumour group p3/h3 in the current study. Moreover, an elevated expression of BIRC5 was found in sample group p2/h2, and a significant down-regulation of both MYCN and BIRC5 was found in group p4/h4 (p < 0.05, Welch t-test). The MNA-specific p3/h3 group was also characterized by a very low expression of NTRK1, whereas the favorable tumour group p1/h1 showed the highest expression of NTRK1. TrkA (or NTRK1) is a well-known marker of favorable NB tumours and its expression has been linked to several cancer forms [56].
In conclusion, by expression profiling of 148 NB tumours from four different Affymetrix-based microarray studies, our data suggest the existence of at least four molecular subgroups of neuroblastoma tumours. Three of the expression-based tumour groups corresponded well to the previously postulated genomic subtypes and a fourth novel group was identified which has not been described elsewhere. The novel tumour group comprised high-stage 11q-deleted tumours with low expression of ALK and MYCN, but high expression of various CNS and nervous system developmental genes. Our findings suggest an alternative classification system based on expression profiling of a 6-gene signature. Further studies of the novel subgroup's specific characteristics are warranted, and will hopefully lead to discoveries on new specific therapeutic targets for children with neuroblastoma.
Materials and methods
Data pre-processing
Raw data files from four published neuroblastoma expression microarray studies generated from two different platforms (i.e. three data sets run on the Affymetrix HU133A platform [22,25,39], and one data set generated from the Affymetrix HGU95Av2 platform [28]) were obtained from ArrayExpress http://www.ebi.ac.uk/microarray-as/ae/ webcite and Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo/ webcite and reanalyzed. The data were pre-processed in three separate groups; i, Data set 1 (De Preter data set) comprising profiles from 23 NB tumours, preamplified and run on the HU133A platform, ii, data set 2 (McArdle and Wilzén data set) comprising 30 neuroblastic tumours run on the HU133A (not preamplified), iii, data set 3 (Wang data set) comprising 101 NB tumours and one brain tissue sample run on the HGU95Av2. Bioconducter for R 2.9.2 (library BioC 2.4) was used to perform gcRMA normalisation on each data set separately [57]. For each probe-set, the maximum expression value over all samples was determined, and probe-sets which showed very low or no detectable expression levels were filtered out (max log2 expression <6). Next, the mean log2 expression level for each Gene symbol was calculated, resulting in 7439 genes for data set 1, 8106 genes for data set 2, and 7542 genes for data set 3.
Principal Components Analysis (PCA)
Principal Components Analysis (PCA) was performed using Omics Explorer 2.0 Beta from Qlucore http://www.qlucore.se webcite on data sets 1 and 2 separately. Using the filtering variance slider, genes with the lowest variance were filtered out until a distinct pattern of groups in the PCA plot appeared, resulting in a set of 414 variables for test group 1 and a set of 716 variables for test group 2 (variance cut-off was approximately 0,4 in both data sets). Next, samples were joined to the nearest neighbour using Euclidean distances of all active samples, and clusters of connected samples were defined as separate PCA sample groups. These were cross-validated by a "leaving-one-out" strategy. The eigenvectors of genes included in the variance-filtered PCA for each data set were sorted according to their loadings in Principal component 1, 2, and 3 (PC1, PC2, and PC3) respectively, and gene lists were compared between the two test groups (Additional file 1).
PubMed gene list
In order to identify known genes which have previously been identified as predictive or differentially expressed in NB disease, the literature was reviewed and gene lists from 15 neuroblastoma expression studies were selected according to the following: 16 differentially expressed genes from Albino et al., 2008 [21], the 55 PCA-based gene module from Asgharzadeh et al., 2006 [15], the 132 PAM classifier gene set from De Preter et al., 2009 [14], 191 differentially expressed genes from De Preter et al., 2006 [22], 220 differentially expressed genes with a fold change above 2 from Fischer et al., 2010 [23], 18 differentially expressed genes from Fischer et al., 2006 [24], the 31 transcripts most strongly associated with the major genetic subtypes of neuroblastoma from McArdle et al., 2004 [25], the 38 top-ranked PAM classifying genes from Oberthuer et al., 2007 [18], the 144 PAM predictor set from Oberthuer et al., 2006 [16], the 41 top-ranked predictive genes from Ohira et al., 2005 [19], the 133 top-ranked genes from Schramm et al., 2005 (from both SAM and PAM analyses) [26], the 89 top-ranked differentially expressed genes from Thorell et al., 2009 [27], 155 differentially expressed genes from Wang et al., 2006 (genes differentially expressed on 1p36 and 11q23, and genes from hierarchical clustering) [28], 72 differentially expressed genes from Warnat et al., 2007 [29], 59 genes selected by data-mining from Vermeulen et al., 2009 [20] (Additional file 4). The intersection of these gene lists resulted in a total number of 1012 unique genes, and among those the genes that occurred in at least two of the 15 gene lists were selected (212 genes, see Additional file 3.). Out of the 212 genes, 157 genes expressed in all three data sets were selected for further co-occurrence search in PubMed. Search terms were the following: "Gene Symbol"[TIAB] AND "Gene expression"[MeSH Terms] AND "neuroblastoma"[MeSH Terms] (search1, Additional file 4), and resulted in hits for 30 genes. In order to get a fair number of PubMed scores we redid the same search including gene alias names for all 30 genes according to Gene cards http://www.genecards.org/ webcite (search 2, Additional file 4). Based on the PubMed results and biological relevance we selected six NB-associated genes from the 30 high score gene list; ALK, BIRC5, CCND1, MYCN, NTRK1 and PHOX2B.
Statistical analysis and subtype discrimination
The frequency of prognostic marker, i.e. INSS stage, outcome, del1p, MNA, del11q, and Gain17q was calculated for each PCA subgroup and tested for significance using Fisher's exact test (table 1). The discriminative power of the six NB-associated genes were tested for significance using a one-way ANOVA test (table 1) and a post hoc (Tukey) test for multiple comparisons. The differential expressions between subgroups were also investigated by Welch t-test (2 sample comparison, unequal variance, table 2). A combined statistic for each gene from the two data sets was constructed as a linear combination of the z-scores (inverse normal distribution of transformed p-values) weighted by the square root of the data sets samples size proportion.
The genomics subtypes were defined based on INSS stage, MNA status, and del11q status (table 4). All tumour cases with MNA were assigned to subtype 2B, all cases displaying del11q with no MNA were assigned to subtype 2A, and all tumours of INSS stage 1, 2, or 3 with no MNA and/or del11q and/or del1p and/or del3p, and which were not dead of disease were assigned to the favourable subtype 1. Tumours that did not fall into any of the categories stated were termed "other" (table 4).
Verification by hierarchical clustering
In order to identify a subgroup discriminative gene set, the 98 most differentially expressed genes between subgroups were identified by SAM. First, the p- group assignments from the two data sets were translated by reassignment into four integrated groups (r1-r4) defined by rules for expression levels of the six NB genes (Additional file 6). Based on these rules, ten samples (three from the DePreter data set and seven from the McArdle/Wilzén data set) could not be assigned to any r-group, which resulted in two sets of 14 and 23 tumours respectively (Additional file 6). A 2 × 2 contingency table shows the r1-r4 representative of the p1-p4 cluster assignment (table 3). From each independent data set the r1-r4 groups were analyzed by SAM using multiclass comparison (i.e. each group was compared to the other groups combined, resulting in four contrasts) [58]. The 4000 most significant genes with a fold change above 2 were selected in each independent data set. Next, SAM gene-lists from the two data sets were compared to create a list of overlapping (or common) genes from each specific contrast. Probe sets with hybridization to more than one gene were filtered out, which resulted in a total of 1987 unique genes overlapping the SAM gene lists from both data sets. For each gene and contrast, the mean log2 fold change from the two data sets was calculated. Next, the genes with the highest combined fold change in each contrast (n = 30) were selected, resulting in a list of 98 unique genes.
The existence of molecular clusters was verified by an unsupervised hierarchical clustering of a third independent data set (Wang data set, comprising 102 samples, [28]). Out of the 98 discriminative genes, 74 genes were present on the Affymetrix HGU95Av2 platform as well as expressed among the 102 samples. Hierarchical clustering of both samples and genes was done using the Average linkage of Euclidian metric (Pearson correlation) for which each variable has been normalized to mean 0 and variance 1. Samples were divided into hierarchical groups based on the dendogram. Samples that allocated in between dendogram trees were assigned to a cluster based on the nearest euclidian neighbour in the PCA-output.
Validation of PCA
In order to verify that the four identified groups could be recognized and discriminated in all three data sets we performed PCA using the same Principal Components loadings. PCA was performed using the R function prcomp on unfiltered expression data, and PCA plots were visualized in 3D using MatLab R2009a. Prior to the analyses, the three pre-processed data sets were filtered to contain the same set of genes (4728 genes in total) and each gene was normalized to center around zero with unit variance.
In the first test, a PCA was performed using the McArdle/Wilzén data (data set 2) and the loading scores from the first three Principal Components were plotted using different colours for each previously identified group (p1-p4 in data sets 1 and 2, and h1-h4 in data set3, see Additional file 7). Next, the loadings from the McArdleWilzén data set were applied to the De Preter and Wang data sets to examine if the same Principal Components (PC1-3) could discriminate the four groups.
In the second test, we repeated the analysis starting with a PCA on the De Preter data set, and the loadings from the De Preter data were then applied to the McArdle/Wilzén and Wang data sets to check if the same pattern appeared (Additional file 7).
Survival analyses by Kaplan Meier
The Overall survival (OS) and Event-free survival (EFS) of patients assigned to the four PCA subgroups (p1-p4) from the two test data sets (De Preter, McArdle/Wilzén) were analysed by Kaplan Meier. OS included totally 43 samples and 4 patients were lost for follow up (3 in p2, and 2 in p3). EFS included totally 35 samples and 11 patients were lost for follow up (5 in p1, 3 in p2, 2 in p3, and 2 in p4). Also, OS and EFS analyses of patients assigned to the four hierarchical subgroups (h1-h4) from the Wang data set were analysed by Kaplan Meier. The OS and EFS analyses included totally 92 samples and 9 patients were lost for follow up (1 in h1, 2 in h3, and 6 in h4). The OS significance was calculated by chi-square Log-rank (Mantel-Cox), and the five year survival significance was calculated by Fisher's exact test.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FA formulated the study design, performed the microarray analysis, PCA, and hierarchical clustering. FA also drafted the manuscript. DD performed programming and cluster calculation, and revised the manuscript. MN verified groups by PCA using unfiltered data. RJ supervised the study design. KD, JV, RS, and JM provided clinical data in terms of status of prognostic marker and survival, and revised the manuscript. SN supervised the study design, statistical analysis, and interpretations of results. All authors read and approved the final manuscript.
This work was supported by grants from the Swedish Medical Council and the Swedish Children's Cancer Foundation.
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