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Mumbaigloss was invited for a tasting at Kurries and Burries this weekend. I was intrigued as it promised to serve pure veg Mexican and Asian food under one roof. Even the Jain version of the dishes were also available. It serves wide variety of cuisines, from Burmese to Thai to South Korean and I wondered what would be the outcome where Mexican and Asian food is being customised to suit the palate of a religious community. Does it affect the taste? Are the prices competitive? So we went in search of the ideal veg Asian-Mex cuisine at this eating joint. Kurries and Burries is a very small café which can hardly seat 20 people at a go. Valet it not available. It is small, and quaint, and there is nothing fancy about it.
We were greeted by Kushal, the young entrepreneur who started this business right after completing his MBA four months ago. His business plan is supported by his family. Most of the employees are his family members working in various roles. The self-service approach makes this little place more of a family run joint. Set in an area that is predominantly populated by Jain community, this small but smart eatery is tucked into one of the by-lanes on Hughes Road, directly opposite a Starbucks joint. "The idea is to make authentic South East Asian and Mexican cuisine available to those who are deprived of them due to religious beliefs. Most of these dishes are originally non-vegetarian, but we have used my mother's recipes to convert them into vegetarian and Jain variants." said Kushal. I was apprehensive as to how the dishes will taste. Usually, when the main ingredients are missing then the chef tends to use overdose of cheese and sauces to make the dishes taste better. I was fearing that and I realised my fears were not baseless to a certain extent.
Though Kushal insisted that the café has been created on QSR (Quick Service Restaurants) and Fine Dine formats but I can safely say that it is more of a QSR and there is no fine dine element here. Use of disposable plates and cutlery and the presentation of the food makes it look more of a fast food joint than a fine dine restaurant. But it has some interesting aspects to it as well like "Beat the Burrito" challenge which requires one to gobble a 1 kg burrito with sides and drinks in under 25 minutes to have the cost waived off and to earn a Facebook post. "About 9 people have taken the challenge, and only 2 have been able to beat it. Time for some serious appetite foodies! Show them what've you got.
The tasting started with Nachos Supremo served with salsa, baked beans and sour cream, all soaked in sinful liquid cheese. It was a regular nachos plate and I can't really say that it was one of the best. The portion size felt a tad bit smaller for a price tag of Rs.150. Mango cocktail was delicious with a generous topping of almond flakes. Crackling Spinach with Paneer costing Rs. 160 didn't leave any impression on me either. Fried spinach heaped on top of paneer with schezwan sauce had way too much oil and was not palatable.
Green Thai Curry stood its ground with delicious thick gravy and a heap of oriental vegetables. The portion size was good too. It was priced at Rs. 210. And finally Mex-wich was served which is their best seller. A bag made of Nachos chips filled with cooked beans, salsa, mozzarella cheese, paneer, and vegetables, all smothered in liquid cheese is given for you to enjoy. Priced at Rs. 150, it is very filling and the cheesy ingredients just make you to go for it again and again.
I would call Kurries and Burries a promising fast food joint. It serves good food but nothing exemplary as the taste is enhanced due to heaps of cheese sauce and salsa. But none the less, it is great for people who want fresh tasty food at value for money.
Will I go there again? Only if I turn pure vegetarian.


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JML Industrie
Specialist in building and commissioning of industrial equipment
Our Group employs today more than 80 peoples. Our know ledge and competence for foundry sand preparation unit (green and core sand) and vibrating machines are recognized in a large part of the world.
Many customers ask us to work with them on complete projects as foundries facilities they are aluminum or ferrous activities.
Our many customers take advantage of our experience to explore the best solutions in terms of investment.
Tomorrow, it could be you.
JML in figures 2018

subset: commoncrawl


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Giant Lego Man Washes Ashore in Holland
A giant 2.5 meter Lego man mysteriously washed up on the Dutch resort of Zandvoort joining the ranks of such publicity stunts as 1984's giant Swatch in Frankfurt and a life-size Lego crane that hit Chile last year. The plastic invader was wearing a shirt with the words, "NO REAL THAN YOU ARE," which makes it unclear if this was even done by Lego.
Workers at a drinks stall rescued the Lego man, with a traditional yellow head and blue torso. "We saw something bobbing about in the sea and we decided to take it out of the water," said a stall worker. A woman nearby added, "I saw the Lego toy floating towards the beach from the direction of England."
Dutch authorities will be taking the man into custody to determine whether he is eligible for asylum status after officials believe he escaped from Denmark's Legoland Billund after suffering from countless human rights violations. "The Lego man appears to be in shock because he hasn't spoken or stopped smiling since his rescue," another official stated.
Crackdown on magic mushrooms
The famously liberal Netherlands has been swinging toward the right, cracking down on immigration, religious freedoms and the freewheeling red light district. The next possible target is magic mushrooms following the death of a 17-year-old French girl, who jumped from a building after eating psychedelic mushrooms while on a school visit.
Regulation of mushrooms is even less stringent than Holland's famously loose laws on marijuana, which is illegal but tolerated in "coffee shops" that are a major tourist attraction. In May, Health Minister Ab Klink ordered the national health institute to perform a new study on the risks of mushrooms.
At a press conference, a spokesman for the national health institute was unable to answer reporters' questions concerning the progress of the study after screaming that a huge Lego man was trying to steal his packet of stroopwaffels.
Flood of complaints against call to ban Koran
Police have received a flood of complaints against a call to ban the Koran made by MP Geert Wilders, leader of the anti-immigration party PVV, last week, reports ANP news service on Wednesday. The exact number of complaints is not yet known but run into 'scores' from around the country, a spokesman for the public prosecution service in Amsterdam said.
The Dutch right-wing politician, known for his views favouring the restriction of immigration, particularly from non-Western countries, his criticism of, and opposition to, Islam, suggested that Muslims should 'tear out half of the Koran if they wished to stay in the Netherlands' because it contained 'terrible things' and that Muhammad would 'in these days be hunted down as a terrorist'.
Dutch liberals are planning on sending Geert Wilders a plate of magic mushrooms and space cakes for his birthday on September 6 in the hope that he lightens the hell up and kills the brain cells responsible for his prejudice.


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Have you ever experienced pain or discomfort but told yourself it will pass and there's no need for a doctor's visit? Hospitals, doctors' offices, and medical tests can be daunting, expensive, and … [Read More...] about Does Acupuncture Really Work?
Main Content
Move Better, Feel Better, Look Better
Albuquerque Acupuncture, Orthopedics, Pain Management, and Sports Medicine
(505) 433-2054
Dr. Geoff Hayes
Acupuncture Clinic in Albuquerque
Conveniently located in Uptown, Endpoint Wellness offers acupuncture in Albuquerque with treatments for pain management, orthopedics and sports medicine, cosmetic acupuncture and facial rejuvenation, and overall wellness.
What Is Acupuncture?
Acupuncture involves the insertion of small, hair-thin needles into specific points of the body called meridians. According to Chinese Medicine, energy flows through these meridians throughout your body, and the placement of needles can adjust this flow of energy and stimulate the body's immune system and boost circulation.
Why Choose Endpoint Wellness?
Dr. Geoffrey N. Hayes, Doctor of Oriental Medicine, is passionate about the role of whole-body wellness in pain management and daily life. Our treatments combine Traditional Chinese Medicine and the modern Western Medicine technique of Microlight therapy to provide pain relief, improve sports performance, boost immune system function and more.
A Full Range of Services
Acupuncture Treatment
Cosmetic Acupuncture
Pain Management
Sports Medicine
Chinese Herbal Formulas
Facial Rejuvenation
Electrical Stimulation
Kinesio Taping
Acupuncture Treatment for Lower Back Pain
As individuals go about their day-to-day activities and progress through life, symptoms of lower back pain become more and more prevalent. In fact, according to the American Chiropractic Association, … [Read More...] about Acupuncture Treatment for Lower Back Pain
Sciatica Pain Management With Acupuncture and more
Ever experienced pain in one side of your lower body extending from your lower back down your thigh and on through the leg? How about pain that stems from prolonged sitting or extended intervals of … [Read More...] about Sciatica Pain Management With Acupuncture and more
Every time I have a visit with Dr. Hayes, I feel relief from my chronic pain issues. He is a very professional and knowledgeable provider. Continue Reading
Amanda L. Endpoint Wellness June 27, 2017
Dr. Geoff Hayes is very caring, versatile, and great to work with. Continue Reading
Kaela P. Endpoint Wellness June 27, 2017
I found that with his treatments I always felt better or had improved mobility right after treatment. Continue Reading
Elaine S. Endpoint Wellness October 12, 2017
I love acupuncture. Dr. Geoff takes his time to inform and explain things. I will come back to him again and again. Continue Reading
Kevin D. Endpoint Wellness May 6, 2017
Frequently Asked Questions
If you've never had acupuncture treatment before, you may have questions about the procedure and what you can expect. Endpoint Wellness is happy to answer these and any other questions you may have.
Does Acupuncture Really Work?
As a holistic treatment for whole-body wellness, acupuncture has been practiced for thousands of years. It is well-respected by the medical community, and treatments are often covered by insurance. Acupuncture is consistently more effective than the placebo effect in scientific trials, and it has even been used successfully in treating pain conditions in animals!
Will Acupuncture Treat My Condition?
The World Health Organization (W.H.O) recommends acupuncture as a treatment for many things, including respiratory diseases, dental pain, orthopedic disorders, neurologic disorders and gastrointestinal disorders. Acupuncture is generally very good at managing the symptoms of chronic and inflammatory conditions. It can improve the immune system and reduce systemic inflammation.
Is Acupuncture Safe?
When performed correctly by a certified Doctor of Oriental Medicine, acupuncture is generally very safe and effective. Unlike pharmaceuticals, it does not have side effects and can be safely used by pregnant women, lactating mothers, patients with compromised immune systems, and others who may not be able to or wish to receive other treatments. When performed with single-use, sterile needles, there is little to no risk of infection or complication from acupuncture treatment.
Does It Hurt?
The needles used in acupuncture are very thin, about the width of a hair. There is little to no discomfort associated with their insertion. During treatment, you may experience a feeling of achiness or pressure spreading through the body; this is a natural effect of the qi manipulation and most patients find it to be relaxing and deeply satisfying.
Move better. Feel better. Look better.
Endpoint Wellness
2730 San Pedro Dr. NE Suite B2
Albuquerque, NM 87110
(505) 433-2054
Endpoint Wellness accepts insurance for acupuncture clients. In order to best serve you, I can verify your insurance benefits before you arrive. Please complete this online form and I will contact you as soon as the benefits are established.


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A small Irish village is mystified by what appears to be a miracle but may actually be murder in the next masterpiece from New York Times-bestselling author Emma Donoghue.
A small village in 1850s rural Ireland is baffled by Anna O'Donnell's fast, which began as a self-inflicted and earnest expression of faith. After weeks of subsisting only on what she calls "manna from heaven," the story of the "miracle" has reached a fever pitch. Tourists flock in droves to the O'Donnell family's modest cabin hoping to witness, and an international journalist is sent to cover the sensational story. Enter Lib, an English nurse trained by Florence Nightingale who is hired to keep watch for two weeks and determine whether or not Anna is a fraud. As Anna deteriorates, Lib finds herself responsible not just for the care of a child, but for getting to the root of why the child may actually be the victim of murder in slow motion.
A magnetic novel written with all the spare and propulsive tension that made Room a huge bestseller, The Wonder works beautifully on many levels—a simple tale of two strangers who will transform each other's lives, a powerful psychological thriller, and a story of love pitted against evil in its many masks.
"It's an intricately, beautifully wrought tale."
"Donoghue's literary prowess creeps like a dark, menacing fog across the pages."
"Claustrophobic, controversial, brilliant . . . inventive, tense, and stringently intelligent."
"Donoghue draws out the narrative suspense with her customary combination of historical verve and emotional delicacy . . . Like Room, this is a thrilling domestic psychodrama that draws its power from quotidian detail as well as gothic horror
"Heartbreaking and transcendent"
"All spectacle and gothic horror and inevitably hard to put down."
"Fresh and unusually lively historical fiction. [Donoghue's] latest novel brings together both her preoccupation with child peril and her gift for history."
"A riveting allegory about the trickle-down effect of trauma."
"With Donoghue's readable prose and lightness of touch, we find ourselves both pausing for thought and racing forward to see what will become of Anna."
Donoghue excels at the microcosm, and her obsessive interest in rooting out the truth makes for a compulsive read. A war is being waged in Anna's tiny bedroom, and its combatants stand for sides not narrowly confined to the Victorian era: science versus faith, progress versus tradition, rich versus poor.
"Room is that rarest of entities, an entirely original work of art. I mean it as the highest possible praise when I tell you that I can't compare it to any other book. Suffice to say that it's potent, darkly beautiful, and revelatory."
"Fodder for endless book club debates."
"Donoghue's superb thriller will keep readers hanging on to every word, pondering how far one will go to prove her faith."
"The Wonder unfolds a cagey, suspenseful stand-off . . . Anna receives the author's full sympathies and is a lively, endearing foil to her incredulous nurse.
"Donoghue is a master of plot, and her prose is especially exquisite in depicting ambiguity. …In The Wonder, Gospel-like in its concern with adversity and hope, miracles are possible–perhaps through God, perhaps through woman."
"A blazing historical novel."
"A fine work, adept and compelling in voice, plot, and moral complexity. . . . Donoghue deals out the cards with real skill."
"Remarkable . . . heartrending. . . . Both gripping and poignant, it's a tribute to human resourcefulness and resilience and extremity, and a stirring portrait of a mother's devotion."
"What at first seems a simple matter becomes far more-stick with it, the payoff is there."
"Beautifully moody with the taut pace of a thriller, The Wonder grapples with the potency of love, both human and divine."
"Thrilling and at moments palm-sweatingly harrowing."
"Her contemporary thriller Room (2010) made the author an international bestseller, but this gripping tale offers a welcome reminder that her historical fiction is equally fine."
"Donoghue is back with a novel as gripping and intense as the popular Room. . . . The prose is so intensely alive, so cinematic that you truly can see the action unfolding before your eyes."
"Donoghue's engrossing novel is loaded with descriptions of period customs and 19th-century Catholic devotional objects and prayers. . . [and] asks daring questions about just how far some might go to prove their faith."
"A masterful, gorgeous, and wickedly well written book.
"Donoghue poses powerful questions about faith and belief"
"Outstanding.... Exploring the nature of faith and trust with heartrending intensity, Donoghue's superb novel will leave few unaffected."
"Readers of historical fiction will gravitate to this tale."
"Riveting. . . . Highly recommended."
"A tense gothic page-turner in which nobody, including the unreliable nurse narrator, are entirely what they seem. A powerful exploration of religion and the sway it holds, The Wonder is equal parts psychological drama and unorthodox love story. A thoroughly enjoyable read from one of the country's premier storytellers.
"If you loved Room, you will love The Wonder. And if there is any justice in Canlit, The Wonder will be on the shortlists of the major literary awards this fall. And after that, undoubtedly the Oscars."
"Fascinating. . . . Like The Turn of the Screw, the novel opens irresistibly, when a young woman with a troubled past gets an enigmatic posting in a remote place. . . . Heartbreaking and transcendent and almost religious in itself."
"Eerie. . . . The reader is seduced by the suspense; compelled to pay as close consideration as the nurse in order to understand precisely what's going on . . . sets a superb pace."
"Sophisticated, provocative, and bracing. "
"A fable as lean and discomfiting as Anna's dwindling body. . . . Donoghue keeps us riveted"
"Threading her stories together like a master weaver, Donoghue takes the reader deeper and deeper into the tapestry of Kissing The Witch. A masterful, gorgeous, and wickedly well written book.
"Donoghue is known for her bestselling novel, Room. . . . [But] she is also well versed in historical fiction. The Wonder brings together the best of all, combining a gracefully tense, young voice with a richly detailed historical setting."
"A haunting novel about good vs. evil."
"Deliciously gothic.... Dark and vivid, with complicated characters, this is a novel that lodges itself deep"


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After long time research I find out accurate causes how do dehumidifier work, So friend
I share a video please see this how to do dehumidifier work. In the time some units have completely different options and effects, all dehumidifiers work in an equivalent basic manner. In short, they pull moisture from the air in your space, lowering wetness levels and making your home safer and additional pleasant. the method works as follows:
A fan in the unit pulls in air from the encompassing space, which generally has high wetness levels
When air passes through the dehumidifier, it touches the cooling coils, that successively pull wetness from the air by lowering the temperature.
Dehumidifier Guy perfectly explain this matter how to do dehumidifier work and dehumidifier reviews and buying guide 2016. This de-moisturized air is then leftover, that then redistributes it into the area – now while not all the wet
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Many models return equipped with a humidistat, which might observe the amount of wetness in the area. this enables you to program the dehumidifier to run till the air hits a particular wetness level so mechanically shut off. the sole different factor to worry concerning is removing water from the reservoir. For dehumidifier reviews visit our reviews page.
Typically, the unit can accompany a plastic bucket – or one thing of an identical nature – that may collect moisture as it is force from the air by the dehumidifier. Some units will also return equipped with a hose hookup. These units permit you to run a hose from the dehumidifier directly to a water drain, guaranteeing that you simply ne'er ought to worry concerning voidance the collected wetness. Most models also have associate degree car shut-off system that may find the water level within the reservoir and shut the process down before it overflows.i over sure to you that we are the provider of best dehumidifier reviews and accurate information, We just help to you that how can you buy a best dehumidifier for your home,office or bathroom.
Take Care of Your Moisture Problem Before It's Too Late
Read this information because dehumidifier guy always provide dehumidifier reviews with essential information.hello dehumidifier guy viewers. its too important to know when you take care your moisture problem. As mentioned earlier having too much wet is unhealthy for your health and for your home. If you watch television shows concerning getting homes like Flip That House on care you will understand that hearing the word wetness may be a massive red flag! that is as a result of wherever there's moisture there's most invariably mildew and it is extraordinarily tough to not possible to urge obviate. All information from my experience.
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Sporting elegance and truly dynamic driving experience couple with superior handling and impressively efficient engines, the BMW 1 Series really is a car that stands out on the road.
Design Features:
Terms and Conditions
The BMW 1 Series 5-door Hatch is available from £299 Advance Payment under the Motability Contract Hire Scheme. Model featured is a BMW 118i SE manual 5-door Hatch starting from £299 on the Motability Contract Hire scheme. All prices are for orders placed and accepted between 1 January 2019 and 31 March 2019 (subject to availability). Models featured may include options at an additional cost. The facilities offered are for the hire (bailment) of goods. The Motability Contract Hire scheme is administered by Motability Operations Limited (Registered Company No. 1373876), City Gate House, 22 Southwark Bridge Road, London, SE1 9HB. To qualify you must be in receipt of the Higher Rate Mobility Component of the Disability Living Allowance, the Enhanced Rate of Mobility Component of Personal Independence Payment (PIP), the War Pensioners' Mobility Supplement (WPMS) or the Armed Forces Independence Payment (AFIP), which will be taken in lieu of the four weekly rental. Terms and conditions apply and are available on request. Test drives are subject to status and availability.


text: Superb place. Do go. You could pick holes - anyone can if they really look hard enough. There are a few little problems with the rooms. But - this is a B & B, and it is easy, because of the location, the rooms, the public spaces to start to imagine you are in a 4 star hotel! It is... More


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13 May – National Volunteer Week Lunch and Learn (Adelaide)
Come and listen to panellists Vanya Bosiocic from Australian Youth Climate Coalition, Alana James from Oaktree and Lucy Camp from Royal Agricultural and Horticultural Society of SA – Royal Adelaide Show discuss their thoughts on – What Volunteering Can Do For You?, in a one hour session facilitated by Volunteer SA&NT.
In particular the session will focus on how volunteering can facilitate experimental learning opportunities, lead to new life skills and challenge conventional thinking. The audience will have an opportunity to ask questions and interact with the panellists whilst learning more about what their organisations do.
Date: Tuesday 13 May
Time: 12pm
Location: City Library – Rundle Place
For more information go to: http://www.volunteeringsa.org.au/VSA_EVENTS_National_Volunteer_Week


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MADRID — Spain's King Juan Carlos has presided over his last military parade before abdicating in favor of his son. Prince Felipe accompanied the king at Sunday's annual Armed Forces day ceremony that honors those who died in the service of the nation.
Also Sunday, a new poll showed most citizens want a referendum to decide whether Spain should remain a monarchy. Pollster Metroscopia found that 62 percent of respondents said they wanted a referendum on the monarchy "at some point." A majority also said they favored the king's abdication. The poll was published Sunday in El Mundo newspaper.
The prince is due to be proclaimed King Felipe VI on June 19.
source: inquirer


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Lapiņa was born in Riga, Latvia, and began her modeling career on March 19, 2005 when she was scouted on street of Riga by Nils Raumanis, the founder of DANDY model management in Riga, Latvia and after placed with MC2 Model Management in New York.Her runway debut was at the Spring 2008 New York Fashion Week in the Benjamin Cho show.In 2008 she signed with model agency Women Management.The main jump in her career in 2008 was the fact that she was chosen for Proenza Schouler exclusive show. Lapiņa has since walked in shows for many notable designers in the Milan, Paris, London, and New York fashion weeks including Shiatzy Chen, Anna Sui, Nina Ricci, Marc Jacobs, Dolce & Gabbana, Prada, Rodarte, Proenza Schouler, Giles, Oscar de la Renta, Diane von Furstenberg, Vivienne Westwood, Miu Miu, Yves Saint Laurent, Carolina Herrera, Bottega Veneta, Versace, Valentino, and Louis Vuitton.
Lapiņa has been photographed in editorials for fashion magazines such as American, German, Russian, Japanese, Espana, Latino America, Italian Vogue magazines, Marie Claire, Numéro, Harper's Bazaar, Dazed & Confused, Love Magazine, W Magazine, and Flair, and has been featured on the cover of Velvet Magazine, Elle France and W magazine Korea. She has been in advertisements for Yves Saint Laurent, NARS, Jill Stuart, Anna Sui, John Galliano, Marc by Marc Jacobs, Sportmax, Uniqlo, Derek Lam, DKNY, Sportmax Fall/Winter 2010 campaign, and in the Miu Miu Fall/Winter 2010 campaign alongside Siri Tollerød, Lindsey Wixson, and Daphne Groeneveld.


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Hi there.
If you are searching for a given GP setting, you probably already hit GPSearch on Azure. In my opinion, this is the best and most valuable ressource for anything related to administrative templates.
And right now, GPSearch is asking for feedback on "why do you need/like/favor it". Given what effort MS puts in evolving GP (almost nothing, to be honest), I encourage all of you readers to give them the feedback they're asking for. This should help to keeping GPSearch online...
Thanks in advance :-)


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Inflammatory breast cancer (IBC) is an aggressive form of BC poorly defined at the molecular level. We compared the molecular portraits of 63 IBC and 134 non-IBC (nIBC) clinical samples.
Genomic imbalances of 49 IBCs and 124 nIBCs were determined using high-resolution array-comparative genomic hybridization, and mRNA expression profiles of 197 samples using whole-genome microarrays. Genomic profiles of IBCs were as heterogeneous as those of nIBCs, and globally relatively close. However, IBCs showed more frequent "complex" patterns and a higher percentage of genes with CNAs per sample. The number of altered regions was similar in both types, although some regions were altered more frequently and/or with higher amplitude in IBCs. Many genes were similarly altered in both types; however, more genes displayed recurrent amplifications in IBCs. The percentage of genes whose mRNA expression correlated with CNAs was similar in both types for the gained genes, but ∼7-fold lower in IBCs for the lost genes. Integrated analysis identified 24 potential candidate IBC-specific genes. Their combined expression accurately distinguished IBCs and nIBCS in an independent validation set, and retained an independent prognostic value in a series of 1,781 nIBCs, reinforcing the hypothesis for a link with IBC aggressiveness. Consistent with the hyperproliferative and invasive phenotype of IBC these genes are notably involved in protein translation, cell cycle, RNA processing and transcription, metabolism, and cell migration.
Citation: Bekhouche I, Finetti P, Adelaïde J, Ferrari A, Tarpin C, Charafe-Jauffret E, et al. (2011) High-Resolution Comparative Genomic Hybridization of Inflammatory Breast Cancer and Identification of Candidate Genes. PLoS ONE 6(2): e16950. https://doi.org/10.1371/journal.pone.0016950
Editor: Francesco Falciani, University of Birmingham, United Kingdom
Received: August 9, 2010; Accepted: January 18, 2011; Published: February 9, 2011
Copyright: © 2011 Bekhouche et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Inserm, Institut Paoli-Calmettes, and grants from Ligue Nationale Contre le Cancer (Label DB) and Institut National du Cancer (ACI2007, AO2008, AO translationnelle 2007 IBC07). IB has been supported by a fellowship from the Algerian Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Inflammatory breast cancer (IBC) [1] is one of the most lethal forms of breast cancer because of its high metastatic potential [2]. IBC is classified T4d in the TNM-UICC classification. Diagnosis is based on clinical signs: edema, erythema, pain, breast widening, and induration [2]. Most cases are associated with a ductal type and a high histological grade [3]. The presence of tumor emboli in dermal lymphatics, although not mandatory for the diagnosis, is a pathological hallmark of 50–75% of IBCs, Emboli are non-adherent cell clusters that rapidly spread by continuous passive dissemination [4], thus favoring both distant metastasis and local recurrence. Despite progresses due to the multi-modality treatment [2], [5], the prognosis remains poor with a 3-year survival of ∼50% [6]. New molecular therapeutic targets need to be identified to improve treatment and increase patient survival.
Molecular mechanisms underlying IBC are poorly known (for review, see [7], [8]). IBCs are more frequently estrogen receptor (ER) and progesterone receptor (PR) negative, ERBB2 and EGFR positive. They frequently present P53 alterations and WISP3 loss-of-expression [9]–[15]. They show high angiogenic and angioinvasive capacities and express high levels of angiogenic factors [16]. They frequently overexpress RHOC [17]–[20], E-Cadherin [18], [21], [22], and NF-κB pathway-related proteins [23], [24]. Recently, a role for eIF4G1 has been suggested in the formation of tumor emboli, pointing to the importance of translation control in IBC [25].
High-throughput molecular approaches have led to important insights in the molecular heterogeneity of non-IBC (nIBC), notably by identifying biologically and clinically relevant subtypes (luminal A and B, basal, ERBB2-overexpressing, normal-like) [26]. More recently, IBCs have been studied at the mRNA level using DNA microarrays [27]–[33]. The results showed the presence of the five molecular subtypes in IBCs, and the definition of IBC versus nIBC gene expression signatures. But the studied series remain relatively small, with 35 IBC samples in the largest one [27], [28]. DNA copy number alterations (CNAs) may account for phenotypic and expression differences between IBCs and nIBCs. However, in contrast to nIBCs [34]–[39], genomic imbalances have not yet been analyzed in IBC by using recent techniques such as array-comparative genomic hybridization (aCGH) or SNP-arrays. The rare genomic studies performed to date used low resolution methods [40]–[42]. Similarly, whole-genome integrated studies (associating analysis of DNA CNAs and mRNA expression levels) have been done in nIBC [34], [39], [43], [44], but never in IBC. Such approaches provide opportunities to better elucidate IBC at the DNA and RNA levels.
Here, we have studied and compared DNA CNAs and mRNA expression deregulation on a whole-genome scale in a large series of IBCs and nIBCs. To our knowledge, this is both the first high-throughput aCGH analysis of IBC and the first whole-genome integrated analysis comparing IBC vs nIBC. This is also the largest series of IBC profiled using high-throughput molecular analyses.
Materials and Methods
Breast cancer samples and histoclinical characteristics
Pre-treatment tumor tissues were collected from 197 patients with invasive adenocarcinomas, who underwent surgical biopsies or initial surgery at the Institut Paoli-Calmettes (IPC, Marseille, France) between 1987 and 2007. Each patient gave written informed consent and the study was approved by the IPC "Comité d'Orientation Stratégique". Tumor samples were macrodissected and frozen in liquid nitrogen within 30 minutes of removal. Before RNA extraction, tumor sections were reviewed by two pathologists (ECJ and JJ) and contained more than 60% of tumor cells. The 197 samples comprised 63 IBCs and 134 nIBCs. IBC tumors were selected upon clinical criteria as T4d tumor. The control group (nIBCs) represented a mixture of early (121 samples, including 68 with pathological axillary lymph node involvement).and locally-advanced stages (13 samples), as found in previous studies [27]–[29], [32], [33]. Using only locally-advanced cases as controls would introduce a bias toward the discovery of molecular differences related to the difference of growth kinetics between IBCs (sudden onset and rapid growth) and nIBCs (long-standing evolution with slower growth). Immunohistochemistry (IHC) status was available on most samples for ER (positivity cut-off: 10%), ERBB2 (0–3+ score, DAKO HercepTest, with >1+ defined as positive), and P53 (positivity cut-off: 1%). Patients were treated according to standard guidelines. The main histoclinical characteristics are listed in Table 1. As expected, IBCs were associated with more unfavorable prognostic features and poorer survival than nIBCs.
DNA and RNA extraction
DNA and RNA were extracted from frozen samples by using guanidium isothiocynanate and cesium chloride gradient. DNA and RNA quality was respectively controlled on polyacrylamide gel electrophoresis and on Agilent Bioanalyzer (Agilent Technologies, Massy, France).
Array-comparative genomic hybridization profiling and data analysis
From the 197 samples analyzed, DNA was available for 173, including 49 IBCs and 124 nIBCs. Genomic imbalances of the DNA samples were determined by using high-resolution 244K CGH microarrays (Hu-244A, Agilent Technologies, Massy, France). A pool of 13 normal male DNA was used as reference. Scanning was done with Agilent Autofocus Dynamic Scanner (G2565BA, Agilent Technologies). Data analysis was done and visualized with CGH Analytics 3.4 software (Agilent Technologies). Extraction of data (log2 ratio) was done from CGH analytics, while normalized and filtered log2 ratio were obtained from "Feature extraction" software (Agilent Technologies). Data generated by probes mapped to X and Y chromosomes were eliminated. The final dataset contained 225,388 unique probes covering 22,509 genes and intergenic regions according to the hg17/NCBI human genome mapping database (build 35). Data were analyzed using circular binary segmentation (CBS) as implemented in the DNA copy R/Bioconductor package [45] with default parameters to translate intensity measurements in regions of equal copy number, each region being defined by at least five consecutive probes. Thus, each probe was assigned a segment value referred to as its "smoothed" value.
We used two different threshold values (log2 ratio >|0.5|, and |1|) to distinguish low level from high level CNAs [43]. DNA copy number patterns were classified as "simplex" (very few CNAs), "complex sawtooth" (many low-level CNAs), or "complex firestorm" (low-level CNAs and recurrent amplifications) [46], [47]. Unsupervised analysis was done with the Cluster program [48] using log2 ratio data, complete linkage and Pearson correlation as similarity metrics. Results were displayed using TreeView [48]. To determine the robustness of the resulting tumor clusters, we used R package Pvclust [49] with multiscale bootstrap resampling using 1,000 iterations. Approximately Unbiased (AU) p-values ≥90% were used to define the significant clusters. To identify altered regions, we used the GISTIC algorithm [50], which computes for each segment through the genome a score based on the frequency of CNA combined with its amplitude, with bootstrapping to calculate the significance level (p<0.05). To identify genes with different CNA frequencies between IBCs and nIBCs, the frequency of CNAs was computed for each probe locus as the proportion of samples showing an aberration therein. Alteration frequencies were compared using the Fisher's exact test and false discovery rate (FDR) was applied to correct the multiple testing hypothesis (p<0.05) [51].
Gene expression profiling and data analysis
Gene expression data of the 197 BCs and 4 normal breast (NB) samples, which represented 1 pool of 4 samples from 4 women, and 3 commercial pools of respectively 1, 2 and 4 normal breast RNA (Clontech, Palo Alto, CA), were quantified using whole-genome DNA microarrays (HG-U133 Plus 2.0, Affymetrix). Scanning was done with Affymetrix GeneArray scanner and quantification with Affymetrix GCOS software. Data were analyzed by the Robust Multichip Average method in R using Bioconductor and associated packages [52]. Robust Multichip Average did background adjustment, quantile normalization, and summarization of 11 oligonucleotides per gene. Before analysis, a first filtering step removed from the dataset the genes with low and poorly measured expression as defined by an expression value inferior to 100 units in all samples. All data was then log2-transformed for display and analysis.
Before clustering analysis, a second filtering retained the 12,813 probe sets with the most variable expression across all samples. Clustering was done with the Cluster program [48] using Pearson correlation as similarity metrics and centroid linkage clustering. Results were displayed using TreeView program [48]. The molecular subtypes of breast cancer were determined using the single sample predictor (SSP) classifier based on the list of 306 intrinsic genes as described [53]. The sample was attributed the subtype corresponding to the most correlated centroid. To develop a predictive model based on the expression of the 24 genes identified by integrated analysis (see below), we applied a logistic regression analysis using the glm function in R statistical package. Once defined, the model was applied to expression data to assign the "IBC-like" or the "nIBC-like" class: first for testing its robustness by leave-one-out cross-validation [54] in the learning set and by validation in an independent set of 24 IBC and nIBC samples, then for estimating its prognostic value in public nIBC datasets.
Genomic and expression data are MIAME-compliant (http://www.mged.org/Workgroups/MIAME/miame.html) and the raw data have been deposited in the MIAME-compliant GEO database (GSE23720).
Integrated analyses of genome and expression data
Before integrated analysis, Affymetrix expression probe sets were matched with Agilent aCGH probes using the hg17/NCBI database. When multiple Affymetrix probe sets mapped to the same gene, the probe sets with an extension « _at », next « s_at », and followed by all other extensions were preferentially kept. When several probe sets with the best extension were available, the one with the highest median value was retained.
We analyzed the degree of correlation between RNA expression and CNAs for the genes and tumors (173 tumors) profiled with both platforms. For each gene with a CNA in at least two IBCs or nIBCs, a Student t-test compared the expression levels in samples with and without the corresponding CNA (FDR-corrected p<0.05). Comparative integrated analysis of IBCs and nIBCs was only applied to the genes with CNA frequencies significantly different between IBCs and nIBCs (628 genes). Genes had to satisfy three other criteria: i) frequencies of combined alterations (gain associated with overexpression vs other combinations, and conversely, loss associated with underexpression vs other combinations) different (Fisher exact test) between IBCs and nIBCs, ii) correlation (Student t-test) between CNA and expression in the 173 samples, and iii) expression different (Student t-test) between IBCs and nIBCs. In the first above-quoted step, overexpression and underexpression for a given gene were assigned using a threshold of |1| corresponding to twice the expression level found in the normal breast pool. These steps are summarized in Figure S1.
Public gene expression data of breast cancer
To test the prognostic performance of our 24-gene signature in nIBCs, we analyzed 6 public data sets collected from five publications [55]–[59], and from the UNC Microarray Database (Table S1). When different publications included the same patients redundancy was eliminated, resulting in 1,781 different patient samples available for analysis. Before analysis, we mapped hybridization probes for the 24 genes across the two oligonucleotide-based platforms used across the series. When multiple probes were mapped to the same GeneID (EntrezGene identification number), the one with the highest variance in a particular dataset was selected to represent the GeneID. Analysis of each data set (using available normalized data) was done separately to guarantee a larger number of genes common with our signature.
Statistical analysis
Correlations between sample groups and histoclinical factors were calculated with the Fisher's exact test for qualitative variables with discrete categories, and the Mann-Whitney test for continuous variables. Follow-up was measured from the date of diagnosis to the date of last news for patients without any event. Disease-free survival (DFS) was calculated from the date of diagnosis until the date of first relapse whatever its location (local, regional or distant) or date of death (when the relapse data was not available) using the Kaplan-Meier method. Survival was compared between groups with the log-rank test. Univariate and multivariate analyses were done using Cox regression analysis. The p-values were based on the Wald test, and patients with one or more missing data were excluded. All statistical tests were two-sided at the 5% level of significance. Analyses were done using the survival package (version 2.30), in the R software (version 2.9.1).
Genome and transcriptome analysis of breast cancer
We first describe the results on the whole set of tumors before addressing the specific question of IBC. High-resolution aCGH was performed on 173 samples, including 49 IBCs and 124 nIBCs. Figure 1A (left) shows the frequency of low level CNAs in all samples. As previously reported [37], [44], the three most frequently gained regions were on 1q, 8q and 17q chromosomal arms, whereas the regions frequently lost were on 8p, 11q and 16q. The median percentage of probe sets displaying a CNA in a sample was 3.5%, with a great variability between samples (range, 0.03–44%). As expected, this percentage was higher in grade 3 tumors (2.1%) than in grade 1 tumors (0.5%; p = 0.005; Mann-Whitney test).
To display the similarity of samples with respect to their genome profiles, unsupervised hierarchical clustering was applied to aCGH data of all 173 samples and all 225,388 oligonucleotide probes (excluding X and Y probes) (Figure 1B). Pvclust algorithm identified three robust tumor clusters, which correlated with the molecular subtypes of samples, and other histoclinical features (Table S2). No correlation existed with the IBC/nIBC status, suggesting similar whole-genome genomic profiles in IBCs and nIBCs, and a similar heterogeneity. Using a previous classification of genome profiles [39], [47], 55 cases (32%) were "simplex", 39 (23%) "complex sawtooth", and 79 (45%) "complex firestorm". This status correlated with the grade of tumors. Only 13% of grade 3 tumors were "simplex", whereas 87% were "complex". Conversely, 61% of grade 1 tumors were "simplex" and 38% "complex".
A) Frequency plots of genome CNA. Frequencies (horizontal axis, from 0 to 100%) are plotted as a function of chromosome location (from 1pter to the top, to 22qter to the bottom), for all breast cancer samples (Global, N = 173), for IBCs (N = 49), and for nIBCs (N = 124). Horizontal lines indicate chromosome boundaries. Positive and negative values indicate frequencies of tumors showing copy number increase and decrease, respectively, with gains (in red) and losses (in green). B) Unsupervised hierarchical clustering of genome CNAs measured for 173 breast cancers on 225,388 probes (without X and Y). Red indicates increased DNA copy number gain and green indicates decreased copy number. The bars to the left indicate chromosome locations ordered like in A). The vertical orange lines define the three significant tumor clusters (I, II and III). Above the dendrogram, p indicates the Approximately Unbiased (AU) p-values defined by Pvclust. Below the dendrogram, the row indicates the clinical type (green for nIBC, and orange for IBC).
The 173 samples and 24 additional samples (197 including 63 IBCs and 134 nIBCs) were profiled using whole-genome mRNA expression microarrays. Figure S2 shows the hierarchical clustering of samples based on the expression of 12,813 probe sets. Samples were sorted into three major clusters, which strongly correlated with histoclinical features (grade, IHC data) and molecular subtypes, but not with the IBC status. As with aCGH analysis, differences due to molecular subtype greatly overcame differences due to clinical type. IBCs were scattered across the three clusters, intermixed with nIBCs, suggesting that, at the RNA level and on a whole-genome scale, they are as heterogeneous as nIBCs.
Regions and genes with significant genome aberrations in IBCs
Our first objective was to establish the first repertoire of genome CNAs in IBC. Table S3 shows the regions with frequent low and/or high level CNAs in the 49 IBCs, defined using the GISTIC algorithm with a significance threshold of p<0.05. Sixty-five regions were gained/amplified. The top ten regions (in term of median GISTIC score) resided on 17q, 6p, 1q, 8q, 11q, 19q, and 8p chromosomal arms. Many of these regions contain genes involved in breast cancer, such as ERBB2, MYC, CCND1 and FGFR1. Thirty-four regions were gained in at least 15% of IBCs. The most frequently gained region (>40%) was 8q12.1–24.3, and contained 461 genes. A total of 216 regions were lost/deleted, with the top ten (in term of median GISTIC score) distributed in 4q, 3q, 1q, 9q, 15q and 2q. Only 4 regions were lost in at least 15% of IBCs.
At the gene level, 321 genes were amplified in at least 10% of IBCs (Table S4). As expected, all were located in the 65 regions gained/amplified reported above. They included validated or potential therapeutic targets such as ANGPT1, ERBB2, FGFR1, GRB7, MYC, PAK1, PNMT, PROSC, SQLE, and STK3. Other targets such as ADAM9 (6% of cases), EGFR, FNTA, and IKBKB (4%), AKT3, AREG, BRAF, EREG, FYN, and RET (2%) were less frequently amplified, but sometimes at a very high level (log2 ratio >|1|). The 15 genes that were the most frequently amplified are located in 17q12 and 8q24 (Table 2). Additionally, rare high-level amplifications targeted other potential therapeutic targets such as DCK, FGF3, FGF10, FLI1, IL6, MFHAS1, ROS1 and SRC. Conversely, 21 genes were deleted in at least 10% of IBCs (Table S4; Table 2), and all were included in the 216 lost/deleted regions.
Comparative genome analysis of IBCs and nIBCs
We compared the genomic profiles of 173 samples (49 IBCs and 124 nIBCs). Globally, as shown in Figure 1A, IBCs looked like nIBCs, since both phenotypes showed similar altered regions with similar frequencies of alterations for most of them. However, some differences were visually apparent, such as the more frequent gain of 1q, 8q and 17q regions in IBCs, or the more frequent loss of 4p, 8p, 11q, and 16q regions in nIBCs. The median percentage of probe sets displaying a CNA for a sample was higher in IBCs (3.7%, range 0.01–14%) than in nIBCs (1.9%, range 0.01–26%; p = 6.1.E–253, Fisher's exact test), even if a great variability between samples existed for both types. IBCs showed a higher proportion of "complex sawtooth" and "complex firestorm" profiles than nIBCs, which conversely showed a higher proportion of "simplex" profiles (p = 0.0009, Fisher's exact test) (Figure S3).
The comparative analysis of regions with frequent low and/or high level CNA in IBCs vs nIBCs showed very similar results in term of number of altered regions (71 and 77 gained respectively, and 210 lost in both types) (Table S5). Many altered regions were similar. Some of them, such as 4q13.2 and 11q11, were similarly altered (similar GISTIC scores) whereas others, such as 1q21.2–q41, 8p11.21–p12, 8q11.1–q24.3, 11q13.33–q14.1, 17q11.1–q21.2, and 20q13.2–q13.33 were differentially altered (different GISTIC scores) either because of a higher frequency and/or higher amplitude of alteration. Other regions were specifically altered, such as 5p15.33 in IBCs or 3q26.1 in nIBCs.
The number of genes amplified or deleted was higher in IBCs than in nIBCs (Table S4). Only 26 genes were amplified in at least 10% of nIBCs, vs 321 in IBCs. As expected, these genes are located within amplicons classically described in breast cancer on 8p, 11q and 17q. Sixteen genes (vs 21 in IBCs) were deleted in at least 10% of nIBCs. Supervised analysis identified 9,148 probes, representing 628 unique sequences/genes, with different CNA frequency in IBCs and nIBCs (Tables S6A–B). A total of 514 genes displayed gains and/or amplifications differentially associated with IBCs vs nIBCs, including 484 genes gained/amplified more frequently in IBCs, and only 30 gained more frequently in nIBCs (Table S6). Compared to nIBCs, IBCs were associated with the gain of 382 genes scattered through 13 chromosomal arms (1p, 4q, 5p, 6p, 6q, 7p, 8q, 10q, 12q, 14q, 17q, 19p, and 20q). Figure S4 shows an example of genomic profiles for the 6q21 region. The frequently gained 8q21.2–24.3 region included more than 230 gained genes, including MTDH (8q22.1), RRM2B, AZIN1, FZD6 (8q22.3), ANGPT1, EIF3E (8q23.1), DCC1, MTBP (8q24.12) and ATAD2, SQLE (8q24.13). By contrast, only 30 genes, scattered through two chromosomal arms (3q and 16p), were more specifically gained in nIBCs, including MYH11, C16orf63 (16p13.11), ERCC4 (16p13.12), and A2BP1 (16p13.3). Thus, specific gains were more frequently observed in IBCs than in nIBCs. The mean frequency of tumors displaying a gain for one gene of these respective lists was 31% for IBCs (maximum 53% for genes located in the 8q22.3–24.11 region), and only 9% for nIBCs (maximum 10% for genes located in the 16p13.11 region). A total of 189 genes were more frequently amplified in IBCs than in nIBCs. Most of them were located within five chromosomal arms (1q, 8p, 8q, 17q, and 18p). Several genes such as RAD21 (8q24.11), MTBP (8q24.12), MYC, PVT1 (8q24.21), ERBB2 (17q12), and CASC3 (17q21.1) are known cancer-related genes. Ninety-nine of these 189 genes were amplified in at least 10% of IBCs. The mean frequency of IBCs with amplification for one of these genes was 14% (maximum 29% for genes such as ERBB2 located in the 17q12 region). In contrast, no gene was more frequently amplified in nIBCs than in IBCs.
A total of 114 genes displayed genomic losses and/or deletions differentially associated with IBCs vs nIBCs, including 68 genes lost/deleted more frequently in IBCs, and 46 more frequently in nIBCs (Table S7). Fifty-nine genes were more specifically lost in IBCs, affecting 7 chromosomal arms (4p, 5q, 6q, 12p, 15q, 19q, and 22q). Examples include EMB (5q11.1), RPS5, UBE2M (19q13.43), SLIT2 (4p15.31) and EZR (6q25.3). Conversely, 46 genes were more specifically lost in nIBCs, affecting two chromosomal arms (8p, and 16q). Examples include PKD1L2 (16q23.2), FOXC2 and FOXF1 (16q24.1). Loss and amplification of BC028701, FKSG2 and KCNU1 (8p12), were correlated with nIBCs and IBCs, respectively. The mean frequency of tumors displaying a loss for one gene of these respective lists was 11% for IBCs (maximum 24% for genes located in the 6q27 and 22q13.1 regions), and 20% for nIBCs (maximum 21% for genes located in the 16q24.1 region). In IBCs homozygous deletions (no copies assuming a modal diploid genome) targeted 9 genes located on five chromosomal arms (3q, 8p, 13q, 14q, and 18q). Four of them were deleted in at least 10% of IBCs. The mean frequency of IBCs with deletion for one of these genes was 10% (maximum 14% for genes located in the 8p23.1 region). No homozygous deletion was more frequently found in nIBCs.
Thus, among the 628 genes (514 gained/amplified and 114 lost/deleted) with CNAs differentially represented between IBCs and nIBCs, 552 (484 and 68; 88%) were associated with IBCs and 76 (30 and 46; 12%) with nIBCs.
Correlations between gene expression and CNA
We compared the degree of CNA-driven mRNA up and downregulation in 49 IBCs and 124 nIBCs profiled on both platforms by analyzing the 13,127 genes common to the two platforms and retained after filtering based on the expression levels.
In IBCs, from all genes with a CNA (gains/amplifications and losses/deletions) in at least two samples, 10.4% of gained genes exhibited mRNA expression correlated with the gain, and 1.5% of lost genes exhibited expression correlated with the loss (Table S8). In nIBCs, these respective features were 10.4% and 9.5%. For the gained genes, the percentages of correlations were similar in IBCs and nIBCS but for the lost genes, this percentage was 7-fold more important in nIBCs than in IBCs (p<1.E-12, Fisher's exact test; OR = 0.14 (CI95% 0.09–0.20)).
Integrated comparative analysis and IBC-specific candidate genes
To identify potential IBC-specific candidate genes, we compared in the 173 IBCs and nIBCs only the genes with deregulated mRNA expression in relation to CNA (Figure S1).
Out of the 628 genes identified by supervised analysis with CNA frequencies different between IBCs and nIBCs, 500 were present on the Affymetrix microarrays. They were represented by 748 probe sets on these microarrays, and 4,259 probes on the Agilent microarrays. From these 500 genes, we identified 24 genes whose expression was deregulated in relation to CNA with significant differences between IBCs and nIBCs (Table 3; Table S9; Figure 2). In all cases, the transcriptional deregulation was associated with IBCs only and corresponded to an overexpression related to a gain (21 genes) and/or amplification (13 genes). By definition, these 24 genes were also overexpressed in IBCs as compared to nIBCs. Twenty of these genes are located in 8q22–q24 and 17q21, including PAPBC1, RAD21, ATAD2, MTSS1, SQLE, ST3GAL1, C17orf37, ABCC3 and PTPN2, previously reported as cancer-related genes. In contrast, no candidate gene was both downregulated and loss-targeted in IBCs.
The 24 candidate oncogenes defined by comparative integrated analysis are shown associated with their corresponding chromosome CNA frequency plot in 49 IBCs and 124 nIBCs. A threshold value of log2 ratio >|0.5| was used to draw chromosome CNA frequency plots using CGH Analytics® software.
To validate the association of these 24 genes with the IBC/nIBC distinction, we analyzed the 24 remaining breast tumors (14 IBCs and 10 nIBCs) only profiled on our Affymetrix platform. None of them had been included in the 173 tumors from which the 24 gene-list had been derived. First, we defined an IBC/nIBC genomic classifier by applying logistic regression to the expression data of the 24 genes in the 173 samples (learning set). As expected, the rate of accurate resulting classification was high (86%). Leave-one-out cross-validation gave similar results with a rate of 82% (p = 2.94E-11, Fisher's exact test; Figure 3A). Then, we applied the predictor to our validation set of 24 samples (Figure 3B): 75% of samples (10/14 IBCs and 8/10 nIBCs; p = 0.03, Fisher's exact test) were correctly classified, suggesting the robustness of the classifier.
A) Classification of the 173 breast cancers (124 nIBCs and 49 IBCs) from which we have generated the 24-gene IBC signature (learning set) by leave-one-out cross-validation. Samples are ordered from left to right according to the decreasing prediction score defined by the 24-gene model. The vertical dashed line indicates the threshold 0 that separates the "IBC-like class" (left of the line) and the "nIBC-like class" (right to the line). Below the curve are some histoclinical and molecular features of the samples: from top to bottom, clinical type (green for nIBC, and orange for IBC), SBR grade (white for grade I, grey for II, and black for III), IHC ER, ERBB2, and P53 status (white for negative, and black for positive). The p-value of correlations between the two tumor classes ("IBC-like" and "nIBC-like) and these features is indicated as follows: ***, <0.001; **, <0.01; *, <0.05; •, <0.1; NS, not significant. B) The classification is validated in the set of 24 independent samples (10 nIBCs and 14 IBCs). C) The same classification method is applied to 1,781 publicly available nIBCs, allowing defining two classes: "IBC-like" and "nIBC-like". D) Kaplan-Meier DFS curves of the two nIBC classes defined in C.
Because of the differences between IBC and nIBC regarding some histoclinical features such as grade, ER, ERBB2 and P53 status, we verified that our 24-gene signature was more associated to the IBC/nIBC distinction than to any of these features. This was done by comparing the p-values testing the correlations between the "IBC-like"/"nIBC-like" classes and each feature (Figure 3A–B). This was further confirmed by regression analysis on the whole set of samples testing and comparing the ability of each variable (24-gene signature, grade, ER, ERBB2 and P53 status) to discriminate IBC from nIBC (Table 4). Three features were significant in univariate analysis (24-gene signature, ERBB2 and P53 status), and two, including the 24-gene signature, remained significant in multivariate analysis. Altogether, these data indicated that our 24-gene signature is more linked to the distinction IBC/niBC than to other potential confounding variables.
Prognostic value of the 24-gene signature
Given the poor prognosis of IBCs, we hypothesized that the 24-gene predictor, if biologically relevant with respect to the IBC/nIBC distinction, might be prognostic in breast cancer. We thus tested its prognostic value in a series of 1,781 clinically annotated nIBCs. Using our 24-gene model (the number of genes common with each data set ranged from 17 to 24), we attributed to each sample an "IBC-like" or "nIBC-like" profile. All series were then pooled, resulting in 338 nIBCs with an "IBC-like" profile and 1,323 with a "nIBC-like" profile (Figure 3C).
We compared the DFS of these two nIBC classes for the 1,420 patients with available clinical outcome. With a median follow-up of 91 months after diagnosis (range, 1 to 220), the "nIBC-like" class had a 5-year DFS of 73%, better than the survival of the "IBC-like" class with a 5-year DFS of 61% (p = 4.4.E-4, log-rank test). Survival curves are shown in Figure 3D.
We then performed univariate and multivariate DFS analyses (Table 5). In addition to the "IBC-like" or "nIBC-like" profile, we tested the variables most frequently annotated in the six data sets: patients' age, pathological tumor size, axillary lymph node status, and grade, and IHC ER and PR status (ERBB2 status not available). All features, except age and PR status, were significant in univariate analysis. The hazard ratio (HR) for relapse was 1.42 for "IBC-like" tumors compared to "nIBC-like" tumors ([95%CI 1.17–1.73], p = 4.7.E-4). In multivariate analysis, these five variables, including the 24-gene model-based classification, retained significant prognostic value, suggesting that our IBC signature is an independent prognostic feature in nIBC, with a HR for relapse for "IBC-like" tumors vs "nIBC-like" tumors equal to 1.34 ([95%CI 1.06–1.70], p = 0.015). This observation confirmed that the 24-gene signature contained some prognostic information, which might explain the worse prognosis of IBCs.
We applied high-throughput molecular analyses to a large series of IBCs and nIBCs. Because copy-number changes drive a considerable proportion of the transcriptional changes [34], we compared genome copy number and expression profiles to identify potential IBC-specific candidate genes. To our knowledge, this is the first high-resolution aCGH study of IBC, the first integrated genomic analysis for IBC vs nIBC comparison, and the largest series of IBCs profiled using high-resolution genomic analytic tools.
Genomic alterations in IBC
Because of the high number of CNAs observed in breast cancer, we used stringent log2 ratio threshold values to define the most specific genomic aberrations. We did not separate IBCs from nIBCs on the basis of whole genome profiles, which were globally very close, suggesting that IBCs are as heterogeneous as nIBCs at the genome level and that different obvious genome alterations are not what distinguish them. However, the number and frequency of CNAs were more important in IBCs than in nIBCs, as well as the proportion of "complex sawtooth" and "complex firestorm" profiles, clearly suggesting that the genomic differences between them are not due to a possible damping of the nIBC signal by higher contamination with normal tissue. Among the 628 genes with CNAs differentially represented between IBCs and nIBCs, 88% were associated with IBCs and only 12% with nIBCs. These results indicate a higher degree of genomic instability in IBCs, in agreement with their high grade, frequency of P53 mutations and their aggressiveness. Given this genomic complexity and heterogeneity of IBCs, and the low degree of differences observed with nIBCs globally, future studies should ideally compare IBCs and nIBCs within molecular subgroups defined by expression (molecular subtypes) and/or by CNA (simplex, complex sawtooth, complex firestorm) profiles as recently suggested [60]. In this context, and given the scarcity of IBC, international collaborations are underway for collecting enough IBC samples that should allow identifying genomic alterations perhaps more specific of IBC.
However, even if the number of profiled samples is relatively small, our series represents the largest tumor set reported in literature, and its study provides several interesting results. We identified 65 regions with gain/amplification and 216 with loss/deletion in IBCs. Few data are available in literature regarding the genomic imbalances in IBCs [40]–[42], and none has been done using high-resolution aCGH. A study of loss of heterozygosity at 71 microsatellite markers in 66 IBCs [41] reported deletions of various chromosomal regions including 3p14–p21.2, 6p, 8p22, 11q22–q23, 11q24–q25, 13q14, and especially 17q21, more frequently than in nIBCs. Our results show some overlap with these results (Table S3). We here identified four 17q21 genes, NSF, ARL17P1, ARL17 and KIAA1267 targeted by loss in 17% of IBCs (Table S3).
In both IBCs and nIBCs the number of regions with CNAs was very close and many altered regions were similar, suggesting that common genes are involved in both types. However, some common regions displayed higher frequency and/or higher amplitude of alteration in IBCs, and other regions were specifically altered in IBCs such as 5p15.33, previously associated with poor prognosis in breast [61] and bladder cancers [62].
At the gene level, the results were similar. Many genes were similarly altered in IBCs and nIBCs in terms of frequency and amplitude of aberrations. However, a larger number of genes displayed recurrent amplifications in IBCs than in nIBCs. Some genes, such as CCND1 and FGFR1, were amplified at the same frequency in the two types, whereas others (99 out of 321 recurrently amplified, 31%), such as ERBB2 and MYC, were more often amplified in IBCs. Of note, all high-level CNAs differentially represented between the two types were associated with IBCs, and none with nIBCs. Several of them code for validated or potential therapeutic targets, which could contribute to enlarge our therapeutic armament in IBC. The difference between IBCs and nIBCs was much less important regarding the genes recurrently deleted. The deletion of the RB1 tumor suppressor gene likely contributes to the genomic instability in IBC.
IBC and transcriptional deregulation
Whole-genome gene expression profiling did not distinguish IBCs and nIBCs more than did aCGH. To our knowledge, the information about the relationship between gene CNAs and mRNA expression in IBC is scarce in the literature. In the present study, we restricted the analysis of expression data to genes with DNA CNA. Having identified the genes with a CNA in at least two IBCs and in at least two nIBCs, we determined those whose mRNA expression correlated with the CNA. In both tumor types, ∼10% of gained genes presented such correlation, in agreement with previous observations obtained with low resolution techniques and less samples in nIBC series [34], [63]. In contrast, this percentage for the genes with loss/deletion was smaller in IBCs (1.4%) than in nIBCs (9.5%), suggesting that epigenetic mechanisms might be more operational in IBCs than in nIBCs.
IBC candidate genes
Integrated analysis of aCGH and expression data identified 24 genes as potential IBC-specific candidate oncogenes, whereas no IBC-specific gene inactivated by loss was found. This does not rule out the likely existence of IBC-specific tumor suppressor genes inactivated by other mechanisms, as well as the existence of a gene expression signatures identified by the sole comparison of whole-genome expression data of IBC vs nIBC. Importantly, the discriminative power of the predictive model - built from the expression levels of these 24 genes - was validated in an independent sample set. Furthermore, this model was an independent prognostic feature in a multicentric series of 1781 nIBCs, indirectly validating its association with IBC, known to be more aggressive than nIBC.
Whether these genes are causative or even predictive of the IBC phenotype in a biological sense or reflect aggressiveness or another associated phenomenon remain to be explored by further in-depth experimental analyses. Several encode proteins involved in the protein translation and transport: MRPL27, a component of mitochondrial ribosomes; VPS13B, involved in vesicle-mediated sorting and transport of proteins within the cell; ABCC3, an ATP-binding cassette transporter, and PABPC1 [64]. Increasing evidence points to a crucial role of translational regulation in cancer development and progression, notably in IBC [65]. PABPC1 is a poly(A)-binding protein (PABP) required for translation initiation. Its interaction with the translation initiation factor eIF4G is crucial for the translational stimulatory effect conferred by the poly(A) tail. In IBC, eIF4G1 reprograms the protein synthetic machinery for specifically increasing the translation of certain mRNAs, notably that encoding p120 catenin, resulting in an increased stabilization of E-cadherin, and that encoding VEGF [66, Silvera, 2009 #2199]]. E-cadherin stabilization maintains the structure of tumor emboli, allowing them to survive and to metastasize as entire structures. VEGF expression accounts for high levels of angiogenesis in IBC and resistance to hypoxia. Our result suggests that PABPC1 could also participate and potentiates this process, allowing IBC cells to adapt to the persistent hypoxia they experience as tumor emboli. Other genes are associated with cell cycle progression: RBM13/MAK16 [67], TAF2, ATAD2, UTP23, MTBP, and DSCC1. TAF2 is a general transcription factor particularly involved in the G2/M transition [68]. ATAD2, as target of E2F, ER and coactivator of MYC [69], links the 3 corresponding pathways, and likely contributes to the aggressiveness of disease through the enhancement of MYC-dependent transcription [70]. UTP23 [71] is a component of the small subunit processome, required for ribosome biogenesis and cell cycle progression at G1. MTBP regulates the E3 ubiquitin ligase activity of MDM2, a critical negative regulator of p53 function [72]. DSCC1 is a component of an alternative replication factor C complex that loads PCNA onto DNA during S phase. Genes of the signature encode proteins involved in RNA processing and transcription: the TAF2 and ATAD2 transcriptional regulators, ARID2, which facilitates ligand-dependent transcriptional activation by nuclear receptors [73], UTP23; INTS2 and INTS8, subunits of the integrator complex, which associates with RNA polymerase II and mediates 3-prime end processing of snRNAs (Baillat, D, Cell 2005). Other genes are associated with metabolism. ST3GAL1 encodes a glycosyltransferase that induces aberrant glycosylation of MUC1 in breast cancer [4], [74]. SQLE encodes a key enzyme of cholesterol biosynthesis, whose expression is associated with poor survival in nIBCs [75]. Two genes, RAD21 and RAD54B, are associated with DNA repair [76]. Finally, four genes (PTPN2, MTSS1, EPN3, and C17ORF37) are associated with cell migration and adhesion and/or poor prognosis of breast cancer. The PTPN2/TC-PTP phosphatase stimulates the ERK pathway [77], and its decreased expression markedly impairs IGF2 induced MCF7 migration [78]. The presence of MTSS1, which encodes for metastasis suppressor 1 [79] appears paradoxal, given its favorable prognostic impact in breast cancer [80]. However, it has been suggested that MTSS1 is unlikely to be a metastasis suppressor, but interacted with RAC, actin and actin-associated proteins to modulate lamellipodia formation [81]. Epsin 3 (EPN3) is involved in extracellular matrix-epithelial cells interactions [82]. C17orf37, whose expression correlates with grade and stage of nIBC [83], promotes invasion and migration of prostate cancer cells by enhancing secretion of uPA, MMP9 and VEGF through NF-kB pathway [84]. Altogether, these different processes (protein translation and transport, cell cycle, RNA processing and transcription, metabolism, cell migration) are consistent with the hyperproliferative and invasive phenotype of IBC. Some of them (protein processing, RNA translation, proliferation and lipid metabolism) have been previously reported as overrepresented among genes or pathways associated with IBC [29], [31].
In conclusion, we report the first description of genomic profiles of IBCs, on a large sample size and with a high-resolution aCGH platform, and the first integrated genomic analysis comparing IBC vs nIBC. This repertoire of whole-genome CNAs in IBCs may serve of basis for further investigations. We show the genomic complexity and heterogeneity of IBCs, which globally look like nIBCs. Many genes targeted by CNA - some of them specific of IBC - have not been previously reported in breast cancer. We have identified 24 IBC-specific potential oncogenes that could explain, at least partially, the IBC phenotype and its aggressiveness, and lead to the development of new therapeutic strategies. As such, they represent new candidates for further clinical and functional validation in IBC. One of them, PABPC1, is particularly interesting as it likely potentiates the role of an alteration recently discovered as essential in IBC pathogenesis. Our findings, as well as the comprehensive database of CNA and mRNA expression generated, constitute a novel step towards the goal of better understanding, and perhaps treating, IBC, even if other alterations of the tumor, as well as those of its microenvironment, at other molecular levels such as DNA mutations, epigenetic regulations, microRNAs [85], proteins and others [4], [25] still need to be identified.
Supporting Information
Figure S1.
Integrated comparative analysis of IBcs and nIBCs with the three successive steps numbered 1, 2 and 3).
Figure S2.
Whole-genome expression profiling of IBCs and nIBCs. A) Hierarchical clustering of 197 samples and 12,813 probe sets with significant variation in mRNA expression level across the samples. Each row of the data matrix represents a gene and each column represents a sample. Expression levels are depicted according to the color scale shown at the bottom. Red and green indicate expression levels respectively above and below the median. The magnitude of deviation from the median is represented by the color saturation. The dendrogram of samples (above matrixes) represents overall similarities in gene expression profiles and is zoomed in B. Colored bars to the right indicate the locations of 6 gene clusters of interest (ECM means extra-cellular matrix). B) Dendrograms of samples. Top, three large groups of tumor samples (designated I to III) are evidenced and delimited by orange vertical lines. Below the dendrogram, are some histoclinical and molecular features of the samples: from top to bottom, clinical type (green for nIBC, and orange for IBC), SBR grade (white for grade I, grey for II, and black for III), IHC ER, ERBB2, and P53 status (white for negative, and black for positive), and intrinsic molecular subtypes (dark blue for luminal A, light blue for luminal B, red for basal, pink for ERBB2-overexpressing, and green for normal-like).
Figure S3.
Proportion of genomic patterns in IBCs and nIBCs.
Figure S4.
6q21 gains are more frequent in IBCs than nIBCs. Profiles of chromosome 6 show higher 6q21 gain frequency in IBC tumors than in nIBC (A). Regional genomic profiles were established with CGH analytics® software (Agilent Technologies), for IBC and nIBC cases (panels B and C) both within the genomic interval [105.9–114.7 Mb] of the long arm of the chromosome 6 (hg17 human genome mapping; build 35 from NCBI). Profiles are distinguishable by different colors corresponding to different cases. Several IBC cases showed 6q21 gain or regional or focused amplification (B), whereas only two nIBC cases displayed a regional amplification (C).
Table S1.
Description of the public nIBC data sets.
Table S2.
Histoclinical correlations of the three aCGH-clustered groups.
Table S3.
Regions with CNAs in IBCs.
Table S4.
Genes with high level CNAs in IBCs and/or nIBCs.
Table S5.
Regions with CNAs in nIBCs.
Table S6.
Genes with copy number gain/amplification frequencies significantly different between IBCs and nIBCs.
Table S7.
Genes with copy number loss/deletion frequencies significantly different between IBCs and nIBCs.
Table S8.
Correlation between DNA CNAs and RNA expression for the 13,127 genes present on both platforms, in IBCs, and in nIBCs.
Table S9.
List of 24 genes with gain or amplification correlated with overexpression showing significant frequency differences between IBCs and nIBCs.
Author Contributions
Conceived and designed the experiments: DB PV MC FB. Performed the experiments: IB PF JA AF GB. Analyzed the data: PF JA AF GB MC FB. Contributed reagents/materials/analysis tools: CT FB PV ECJ CC GH JJ. Wrote the paper: MC FB.
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text:
What's in a Name?
Bike racing in the UK and the rest of Europe was always traditionally a working class sport, certainly up until the 1950s.
Working class people, for example your average factory worker would use a bicycle as transport to and from work each day.
It was natural that some of them would be into bike racing. Frames for racing bikes were often built by a local framebuilder. Many of these framebuilders were ex bike racers themselves. Well-known personalities who put their first and last name on the frames they built.
Names like Bob Jackson, Harry Quinn, and Freddie Grubb. Notice the first names were always abbreviated in true working class tradition. The name on the frames did not read Robert Jackson, Harold Quinn, or Frederick Grubb, that would have been far too grandiose for their working class customers to relate.
So quite naturally I followed suit when I started building frames and put 'dave moulton' on the frame, all in lower case letters which made it somewhat distinctive. Being working class myself, I was always known as "Dave." Only my mother called me David.
I came to the US in 1979, and two years later in 1981 I was in Southern California, building frames under my own name again. I was essentially starting over again from scratch. The general populous did not have a clue who Dave Moulton was, only a handful of people in the bike business knew of my work.
There was an immediate resistance to the name. I got comments like, "Why do you put your first and last name on there, people will think it is my name." "My name," meaning of course the customer. "Not exotic sounding enough," was another comment I heard all the time.
I even had requests to build frames without my name on it. I stood my ground there, and refused those orders, and in a relatively short time my reputation as a framebuilder grew, and the resistance to the name disappeared.
However, I was reminded of this just last week. For this last year I have been designing cycling related tee shirts. I found bike tees are often lacking cleaver design and sometimes quite lame when it comes to any kind of a message. I am trying to come up with interesting and different designs. Conversation pieces if you will.
So when I recently had a request for my four "m" logo on a shirt. I thought it might be an idea to add something extra to the design.
Although I am a little more well-known in the US than I was back in the 1980s, probably only a fraction of one percent of the general population would have even heard of Dave Moulton, much less be familiar with my logo.
So put a large four "m" logo on a shirt, and occasionally someone will recognize it as Dave Moulton's logo. Everyone else will not even give it a second look, because to them it is a meaningless symbol. My thought was to combine the logo with a design based on something called a "Celtic Knot." (See top picture.)
Someone might say to the wearer, "That's a cool design on your shirt, does it have a meaning?" Which is what I mean by a conversation piece. Others in the know, might give the shirt a second look, then realize, "That's the Dave Moulton logo in there."
But when I asked for feedback on the Dave Moulton Bikes Facebook page, the pure fans of my bikes hated it. "Too busy." And, "Takes away from the simplistic beauty of the original logo." Were typical comments.
My thoughts were. I'm no longer selling bikes. My logo does not have to jump out and grab you by the throat. It can be a little more subtle. But on the other hand, this is a tee shirt with my logo on it. Who else will it appeal to except fans of my bikes?
Oh well, back to the drawing board.


text:
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The Dell DR4100 disk backup appliance provides in-line deduplication, compression, and deduplicated replication capabilities designed to optimize storage utilization. The Dell DR4100 also offers impressive scalability with support for up to 27TB of physical capacity and up to 405TB of logical capacity per system.
Enhanced Data Protection.
Delivering many-to-one replication capabilities, Dell's DR4100 backup and recovery appliance offers cost-effective and reliable data protection for multiple remote sites. Featuring support for multiple backup workloads, enhanced data protection is provided through encrypted and compressed replication between the DR4000 and the DR4100.
Simplified Management.
Featuring broad, integrated software support, the Dell DR4100 supports already-in-place backup software applications. Additionally, the Dell DR4100 disk backup appliance delivers easy management and monitoring by means of a simple, intuitive dashboard management interface.


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Latest update for Google Glass adds photo and video backup, and more
Another update is coming for Google Glass, which will see photo and and video backup, as well as the ability to remove photos and videos from your timeline, and smarter phone answering.
Backing up photos and videos only takes place when Glass is on charge, and connected to your Wi-Fi network. If you want to force a backup, this is now possible, where you simply swipe up to the Auto Backup card in Settings and tap it to begin backing your Glass up. Clearing videos and photos from your timeline, according to Google its: "one of our top community requests". Clearing all of the photos and videos from your timeline can be done by tapping the Auto Backup card, and then swiping up forward.
The last part of the update is to Glass being smarter at answering phone calls. If you answer the phone call with your smartphone, the smartphone will take over the audio, if you answer on Glass, the Glass will be responsible for audio. If Glass is left inactive (such as when it detects it's not on your face) then phone calls won't be blasted to it.


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Emails from real students
Subject: i passed the exam
Date: October 30, 2012 11:32:55 AM EDT
just wanted to let you know..I took it yesterday...Thanks Ashini
Date: Tue, Aug 28, 2012 at 2:37 PM
Subject: Thanks
Hi Jatinder,
I passed the PMP exam.
Thanks to your materials and PMP questions. It helped a lot.
Date: Fri, May 18, 2012 at 11:29 AM
Subject: Re: Current course is officially over
I first want to thank you for putting a great course together. I am sorry I was not able to take advantage of this wonderful resource because of time constraints of new job. I would like to join soon again with another group. When I can fully utilize and be a good group participant. I would like to be a testimonial for you also. Please let me know how I can help you. I appreciate your work and consideration.
Best regards
Terry Chavous