Posts Tagged ‘oncology’


Reporter: Stephen J. Williams, Ph.D.


3.3.8   The 3rd STATONC Annual Symposium, April 25-27, 2019, Hilton Hartford, CT, 315 Trumbull St., Hartford, CT 06103, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair


The three-day symposium aims to bring oncologists and statisticians together to share new research, discuss novel ideas, ask questions and provide solutions for cancer clinical trials. In the era of big data, precision medicine, and genomics and immune-based oncology, it is crucial to provide a platform for interdisciplinary dialogues among clinical and quantitative scientists. The Stat4Onc Annual Symposium serves as a venue for oncologists and statisticians to communicate their views on trial design and conduct, drug development, and translations to patient care. To be discussed includes big data and genomics for oncology clinical trials, novel dose-finding designs, drug combinations, immune oncology clinical trials, and umbrella/basket oncology trials. An important aspect of Stat4Onc is the participation of researchers across academia, industry, and regulatory agency.

Meeting Agenda will be announced coming soon. For Updated Agenda and Program Speakers please CLICK HERE

The registration of the symposium is via NESS Society PayPal. Click here to register.

Other  2019 Conference Announcement Posts on this Open Access Journal Include:

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Cell Therapy Market to Grow Beyond Oncology As Big Pharma Expands Investments

Reporter: Irina Robu, PhD

Collaborations of Big Pharma with small to mid-segment companies are currently focusing R&D on precision medicine. The market is valued at $2.70 billion in 2017 and is expected to reach $8.21 billion in 2025. A varied therapeutic focus and implementation of advanced manufacturing technologies such as single-use bioreactors, will pave a way for unique cell-gene and stem cell – gene combination therapies.
Novartis and Gilead are the first companies to adopt pay for performance business for their CAR-T cell therapies. In addition to innovative pricing models, Pharma companies are also showing a preference for risk sharing and fast-to-market models in order to support the development of novel therapies. Moreover, developments in cell culturing techniques alongside the use of different stem cells such as adipose-derived stem cells, mesenchymal stem cells, and induced pluripotent stem cell will reinforce the market with superior treatment options for non-oncological conditions such as neurological, musculoskeletal, and dermatological conditions.

With the high request for cell therapies, numerous growth opportunities can occur such as:

  • With more than 959 ongoing regenerative medicine clinical trials, the market finds opportunity across both stem cell and non-stem cell-based therapies.
  • Curative combination therapies which help find application in identifying the right patient as well as predicting the immune response in cancer patients.
  • Implementation of IT solutions and single-use manufacturing techniques for optimizing small-volume, high-value manufacturing of novel cell therapies, thus dropping the time to market radically.
  • Emerging Business Models which aid market players focus on academic and research collaborations together with industry collaborations to support therapeutic and technological innovations.




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City of Hope, Duarte, California – Combining Science with Soul to Create Miracles at a Comprehensive Cancer Center designated by the National Cancer InstituteAn Interview with the Provost and Chief Scientific Officer of City of Hope, Steven T. Rosen, M.D.

Author: Gail S. Thornton, M.A.

Co-Editor: The VOICES of Patients, Hospital CEOs, HealthCare Providers, Caregivers and Families: Personal Experience with Critical Care and Invasive Medical Procedures


City of Hope (https://www.cityofhope.org/homepage), a world leader in the research and treatment of cancer, diabetes, and other serious diseases, is an independent, biomedical research institution and comprehensive cancer center committed to researching, treating and preventing cancer, with an equal commitment to curing and preventing diabetes and other life-threatening diseases. Founded in 1913, City of Hope is one of only 47 comprehensive cancer centers in the nation, as designated by the National Cancer Institute.

City of Hope possesses flexibility that larger institutions typically lack. Innovative concepts move quickly from the laboratory to patient trials — and then to market, where they benefit patients around the world.

As a founding member of the National Comprehensive Cancer Network, their research and treatment protocols advance care throughout the nation. They are also part of ORIEN (Oncology Research Information Exchange Network), the world’s largest cancer research collaboration devoted to precision medicine. And they continue to receive the highest level of accreditation by the American College of Surgeons Commission on Cancer for their exceptional level of cancer care.

As an innovator, City of Hope is a pioneer in bone marrow and stem cell transplants with one of the largest and most successful of its kind in the world. Other examples of its leadership and innovation include,

  • Numerous breakthrough cancer drugs, including Herceptin, Rituxan, Erbitux, and Avastin, are based on technology pioneered by City of Hope and are saving lives worldwide.
  • To date, City of Hope surgeons have performed more than 10,000 robotic procedures for prostate, kidney, colon, liver, bladder, gynecologic, oral and other cancers.
  • They are a national leader in islet cell transplantation, which has the potential to reverse type 1 diabetes, and also provide islet cells for research at other institutions throughout the U.S.
  • Millions of people with diabetes benefit from synthetic human insulin, developed through research conducted at City of Hope.
  • Their scientists are pioneering the application of blood stem cell transplants to treat patients with HIV- and AIDS related lymphoma. Using a new form of gene therapy, their researchers achieved the first long-term persistence of anti-HIV genes in patients with AIDS-related lymphoma — a treatment that may ultimately cure lymphoma and HIV/AIDS.


Additionally, City of Hope has three on-campus manufacturing facilities producing biologic and chemical compounds to good manufacturing practice (GMP) standards.

City of Hope launched its Alpha Clinic, thanks to an $8 million, five-year grant from the California Institute for Regenerative Medicine (CIRM). The award is part of CIRM’s Alpha Stem Cell Clinics program, which aims to create one-stop centers for clinical trials focused on stem cell treatments for currently incurable diseases. The Alpha Clinics Network is already running 35 different clinical trials involving hundreds of patients, 17 of which are being conducted at City of Hope. Current clinical trials include transplants of blood stem cells modified to treat patients with AIDS and lymphoma, neural stem cells to deliver drugs directly to cancers hiding in the brain, and T cell immunotherapy trials.

Located just northeast of Los Angeles, landscaped gardens and open spaces surround City of Hope’s leading-edge medical and research facilities at its main campus in Duarte, California. City of Hope also has 14 community practice clinics throughout Southern California.

COH robotic (1)COH Helford H (1)COH1 Dr__Rosen_Clinic-2 (2)COH8 Janice_Huss-7COH7 COH_1369COH6 GMP_0454COH4 DSC_9279

Image SOURCE: Photographs courtesy of City of Hope, Duarte, California. Interior and exterior photos of the City of Hope, including Dr. Steven T. Rosen and his team.


Below is my interview with the Provost and Chief Scientific Officer of City of Hope, Steven T. Rosen, M.D., which occurred in April, 2017.


What sets City of Hope apart from other hospitals and research centers?

Dr. Rosen: City of Hope offers a unique blend of compassionate care and research innovation that simply can’t be found anywhere else.

We’re more than a medical center, and more than a research facility. We take the most compassionate patient-focused care available, combine it with today’s leading-edge medical advances, and infuse both with a quest to deliver better outcomes.

I’m proud to say that we’re known for rapidly translating scientific research into new treatments and cures, and that our technology has led to the development of four of the most widely used cancer-fighting drugs, Herceptin (trastuzumab), Avastin (bevacizumab), Erbitux (cetuximab), and Rituxin (rituximab).

City of Hope is a family. Our special team of experts treats the whole person and the family, not just a body, or a case or a disease. In fact, some of our patients have shared their stories of success. It is gratifying for me and our many health professionals to be able to make a positive difference in their lives.

Eleven years ago, Los Angeles firefighter Gus Perez was facing a battle far greater than any he’d ever known. He was diagnosed with CML (chronic myelogenous leukemia). Gus began receiving the drug Gleevec, which put him into remission. Given the drug’s success, he almost resigned himself to staying on it, yet was drawn to another option: undergoing a bone marrow transplant at City of Hope. “I went to my favorite ocean spot,” Gus recalls. “I put on my wetsuit, like I’ve done thousands of times, and paddled out. Every wave was special because I wasn’t sure if I was ever going to be back. And I remember getting out of the water and counting the steps to my car, thinking, ‘I’m going to beat this. I’m going to retrace those steps.’ And I’m happy to say I was able to do it.” Gus and his family recently celebrated the 10th anniversary of his bone marrow transplant. “City of Hope is more than just medical treatment,” Gus says. “They have to put you back together from the ground up. And to me, that’s truly a miracle.”


As an active 14-year-old, Nicole Schulz loved cheerleading and hanging out with her friends. Then her whole world changed. Nicole learned that her fatigue and other symptoms weren’t “just the flu,” but the effects of acute myelogenous leukemia (AML), an aggressive disease that rendered her bone marrow 97 percent cancerous. Nicole spent the next three and a half months at City of Hope, fighting the cancer with a daily regimen of chemotherapy and blood and platelet transfusions. “It put me into remission,” Nicole says. “But I wasn’t cured. And I wanted a cure.” Fortunately, Nicole was a candidate for a bone marrow transplant. Her malfunctioning marrow cells would be replaced with healthy marrow from a matching unrelated donor. “I never gave up — and neither did City of Hope,” Nicole says. After two bone marrow transplants and tremendous perseverance, Nicole is back to living the life she once knew and quickly making up for lost time.


When Jim Murphy’s doctor called and asked to see him on Christmas Eve, Jim knew it wasn’t going to be good news. And he was right. “The diagnosis was esophageal cancer,” Jim says. “Once they tell you that, there’s nothing you can do but formulate your action plan.” Jim would need to undergo chemotherapy, radiation and surgery to remove the tumor from his esophagus. It would require taking two-thirds of his esophagus and a third of his stomach. Despite the intense treatment, Jim was determined to keep his life as normal as possible. Throughout his chemotherapy and radiation therapy, he never missed a day of work, even riding his mountain bike to and from City of Hope to take his treatments. “I needed to show myself one victory after another,” Jim says. “I know City of Hope appreciated the fact that I was fighting as hard as they were.” Now cancer-free for several years, Jim credits City of Hope with giving him the best chance to fight his disease. “What really impressed me was that the research was right there at City of Hope. If they have something experimental, it goes from the researcher, right to the doctor and right to you. It’s the ultimate weapon — doctors reaching out for researchers, researchers reaching out for doctors. And the patient wins.”


City of Hope is a pioneer in the fields of bone marrow transplantation, diabetes and breakthrough cancer drugs based on technology developed at the institution.  How are you transforming the future of health care by turning science into a practical benefit for patients? 

Dr. Rosen: This is a distinctive place where brilliant research moves rapidly from concept to cure. That’s what we do—we speed breakthroughs in the lab to benefit patients in the clinic

Many know us for our leadership in fighting cancer, but fighting cancer is only part of our story. For decades, we’ve been making history in the fight against diabetes and other life-threatening illnesses that can be just as dangerous, and shattering, to patients and their families.

Every year, we conduct 400+ clinical trials, enrolling 6,000+ patients; hold 300+ patents and submit nearly 30 applications to the U.S. Food and Drug Administration (FDA) for investigational new drugs; and offer comprehensive assistance for patients and their families, including patient education, support groups, social resources, mind-body therapies and patient navigators.

We also translate breakthrough laboratory findings into real, lifesaving treatments and cures, and manufacture them at three on-campus facilities. Our goal is to get patients the treatments they need as fast as humanly possible.

We are in the race to save lives – and win. In our research efforts, we are teaching immune cells to attack tumors and Don J. Diamond [Ph.D.], Vincent Chung, [M.D.], and other City of Hope researchers launched a clinical trial seeking ways to effectively activate a patient’s own immune system to fight his or her cancer. The team is combining an immune-boosting vaccine with a drug that inhibits tumor cells’ ability to grow — to encourage immune cells to attack and eliminate tumors such as non-small cell lung cancer, melanoma, triple-negative breast cancer, renal cell carcinoma and many other cancer types.

City of Hope’s Diabetes & Metabolism Research Institute is committed to developing a cure for type 1 diabetes (T1D) within six years, fueled by a $50 million funding program led by the Wanek family. Research is already underway to unlock the immune system’s role in diabetes, including T cell modulation and stem cell-based therapies that may reverse the autoimmune attack on islet cells in the pancreas, which is the cause of T1D. City of Hope’s Bart Roep [Ph.D.], previously worked at Leiden University Medical Center in the Netherlands, where he was instrumental in launching a phase 1 clinical trial for a vaccine that aims to spur the immune system to fight, and possibly cure, T1D. Plans are developing for a larger, phase 2 trial to launch in the future at City of Hope.


What makes your recent alliance with Translational Genomics Research Institute (TGen) different from other efforts in precision medicine around the country and within our Government to identify treatments for cancer?

Dr. Rosen: Precision medicine is the future of cancer care. Since former Vice President’s Joe Biden’s Moonshot Cancer program was launched to achieve 10 years of progress in preventing, diagnosing and treating cancer, within five years, federal cancer funding has been prioritized to address these aims.

City of Hope and the Translational Genomics Research Institute (TGen) have formed an alliance to fast-track the future of precision medicine for patients. Our clinical leadership as a comprehensive cancer center combined with TGen’s leadership in molecular cancer research will propel us to the forefront of precision medicine and is further evidence of our momentum in transforming the future of health.

In fact, most recently scientists at TGen have identified a potent compound in the fight for an improved treatment against glioblastoma multiforme (GBM), the most common and deadly type of adult brain cancer. This research could represent a breakthrough for us to find an effective long-term treatment. The compound prevents glioblastoma from spreading, and leaves cancer vulnerable to chemotherapy and radiation.  Aurintricarboxylic Acid (ATA) is a chemical compound that in laboratory tests was shown to block the chemical cascade that otherwise allows glioblastoma cells to invade normal brain tissue and resist both chemo and radiation therapy.

The goal is to accelerate the speed at which we advance research discoveries into the clinic to benefit patients worldwide.


As a prestigious Comprehensive Cancer Center, City of Hope was named this year as one of the top 20 cancer centers for the past 10 years. How do you achieve that designation year after year? And what specific collaborations, clinical trials and multidisciplinary research programs are under way that offer benefits to patients?

Dr. Rosen: It’s simple – we achieve this through the compassion, commitment and excellence of the City of Hope family, which includes our world-class physicians, staff, supporters and donors.

We look to find the best and brightest professionals and bring them to City of Hope to work with our amazing staff on research, treatments and cures that not only change people’s lives, but also change the world.

We also have a community of forward-looking, incredibly generous and deeply committed supporters and donors. People who get it. People who share our vision. People who take their capacity for business success and apply it to helping others. They provide the fuel that drives us forward, enabling us to do great things.

City of Hope has a long track record of research breakthroughs and is constantly working to turn novel scientific research into the most advanced medical services.

Right now, we have a number of collaborative programs underway, including: Our alliance with TGen to make precision medicine a reality for patients, The Wanek Family Project to Cure Type 1 Diabetes, and Immunotherapy and CAR-T cell therapy clinical trials, which aim to fight against brain tumors and blood cancers.

More specifically, our research team led by Hua Yu, [Ph.D.] and Andreas Herrmann, [Ph.D.], developed a drug to address the way in which cancer uses the STAT3 protein to “corrupt” the immune system. The drug, CpG-STAT3 siRNA, halts the protein’s ability to “talk” to the immune system. It blocks cancer cell growth while sending a message to surrounding immune cells to destroy a tumor, and it may also enhance the effectiveness of other immunotherapies, such as T-cell therapy.

We could also see a functional cure for HIV in the next 5 to 10 years. Gene therapy pioneer, John A. Zaia, [M.D.], the Aaron D. Miller and Edith Miller Chair in Gene Therapy, the director of the Center for Gene Therapy within City of Hope’s Hematologic Malignancies and Stem Cell Transplantation Institute, as well as principal director of our Alpha Clinic, and researchers are building on knowledge gained from the case of the so-called “Berlin patient” whose HIV infection vanished after receiving a stem cell transplant for treatment of leukemia. The donor’s CCR5 gene, HIV’s typical pathway into the body, had a mutation that blocked the virus. The team launched a clinical trial that used a zinc finger nuclease to “cut out” the CCR5 gene, leaving HIV with no place to go. Their goal: to someday deliver a one-time treatment that produces a lifetime change. Integral to the first-in-human trials are the nurses who understand the study protocols, potential side effects and symptoms.


Would you share some of the current science under way on breakthrough cures for cancer?

Dr. Rosen: We are achieving promising results in many innovative approaches – gene therapy, targeted therapy, immunotherapy and all aspects of precision medicine. We are also forging new partnerships and collaboration agreements around the world.

Let me share with you a few examples of our cutting-edge science.

City of Hope researchers identified a promising new strategy for dealing with PDAC, an aggressive form of pancreatic cancer. The bacterial-based therapy homes to tumors and provokes an extremely effective tumor-killing response.

Teams at City of Hope are working to load nanoparticles with small snippets of DNA molecules that can stimulate the immune system to attack tumor cells in the brain. This innovative approach can overcome the blood-brain barrier, which blocks many drugs from reaching the tumor site.

A pioneer in islet cell transplantation for the treatment of diabetes, City of Hope conducted a clinical trial to refine its transplantation protocol. Because this new protocol includes an ATG (antithymoglobulin) induction, the immune system will not harm the transplant. The immune-suppression strategy used in the trial is considered a significant improvement over the protocol used in previous islet cell transplant trials.

City of Hope physicians and scientists joined a multinational team in reporting the success of a phase II clinical trial of a novel drug against essential thrombocythemia (ET). ET patients make too many platelets (cells essential for blood clotting), which puts them at risk for abnormal clotting and bleeding. All 18 patients treated with the drug, imetelstat, exhibited decreased platelet levels, and 16 showed normalized blood cell counts.

Researchers found that the CMVPepVax vaccine — developed at City of Hope to boost cellular immunity against cytomegalovirus (CMV) — is safe and effective in stem cell transplant recipients. Building on this discovery, City of Hope and Fortress Biotech formed a company to develop two vaccines, PepVax and Triplex, against CMV, a life-threatening illness in people who have weakened or underdeveloped immune systems such as cancer patients and developing fetuses. The vaccines are the subjects of multisite clinical trials. These City of Hope vaccines could open the door to a new way of protecting cancer patients from CMV, a devastating infection that affects hundreds of thousands of people worldwide.


In what ways does the initial vision of Samuel H. Golter impact the work you are doing today? What does the tagline – “The Miracle of Science with Soul” – mean?

Dr. Rosen: 100+ years ago, Samuel Golter, one of the founders of City of Hope said: “There is no profit in curing the body if in the process we destroy the soul.” For decades, City of Hope has lived by this credo, providing a comprehensive, compassionate and research-based treatment approach.

“The Miracle of Science with Soul” refers to the lives that we save by uniting science and research with compassionate care.

“Miracle” represents what people with cancer and other deadly diseases say they want most of all.

“Science” speaks to the many innovations we’ve pioneered, which demonstrate that medical miracles happen here.

“Soul” represents our compassionate care. We’re an untraditional health system — and our people, culture and campus reflect this.


Can you please describe how City of Hope has evolved throughout its 100-year history from a tuberculosis sanitorium into a world-class research-centered institution? 

Dr. Rosen: City of Hope is a leading comprehensive cancer center and independent biomedical research institution. Over the years, our discoveries have changed the lives of millions of patients around the world.

We pioneered the research leading to the first synthetic insulin and the technology behind numerous cancer-fighting drugs, including Herceptin (trastuzumab), Avasatin (bevacizumab), Erbitux (cetuximab), and Rituxin (rituximab).

As previously mentioned, we hold 300+ patents, have numerous potential therapies in the pipeline at any given time, and treat 1,000+ patients a year in therapeutic clinical trials.

These numbers reflect our commitment to innovation and rapid translation of science into therapies to benefit patients.

We are home to Beckman Research Institute of City of Hope, the first of only five Beckman Research Institutes established by funding from the Arnold and Mabel Beckman Foundation. It is responsible for fundamentally expanding the world’s understanding of how biology affects diseases such as cancer, HIV/AIDS and diabetes.

Recognizing our team’s accomplishments in cancer research, treatment, patient care, education and prevention, the National Cancer Institute has designated City of Hope as a comprehensive cancer center. This is an honor reserved for only 47 institutions nationwide. Our five Cancer Center Research Programs run the gamut from basic and translational studies, to Phase I and II clinical protocols and follow-up studies in survivorship and symptom management.

City of Hope’s Diabetes & Metabolism Research Institute offers a broad diabetes and endocrinology program combining groundbreaking research, unique treatments and comprehensive education to help people with diabetes and other endocrine diseases live longer, better lives.

Our dedicated, multidisciplinary team of healthcare professionals at the Hematologic Malignancies & Stem Cell Institute combine innovative research discoveries with superior clinical treatments to improve outcomes for patients with hematologic cancers.

Working closely with the City of Hope comprehensive cancer center’s Developmental Cancer Therapeutics Program and other cancer centers, the Medical Oncology & Therapeutics Research multidisciplinary program includes basic, translational and clinical research and fosters collaborations among scientists and clinicians.

City of Hope’s Radiation Oncology Department is on the forefront of improving patient care, and our staff is constantly studying new research technologies, clinical trials and treatment methods that can lead to better outcomes and quality of life for our patients.

What attracted you to City of Hope? And how do you define success in your present role as provost and CSO?

Dr. Rosen: Helping cancer patients and their families gives me a sense of purpose. I encourage everyone to find a passion and find an organization that fits their passion. City of Hope is a special place. What we do is bigger than ourselves.

I define success as finding cures and helping patients live stronger, better lives. I am focused on leading a diverse team of scientists, clinicians and administrative leaders committed to discovering breakthroughs and specialized therapies.

COH2 Dr__Steve_Rosen_

Image SOURCE: Photograph of Provost and Chief Scientific Officer Steven T. Rosen, M.D., courtesy of City of Hope, Duarte, California.


Steven T. Rosen, M.D.
Provost and Chief Scientific Officer

City of Hope
Duarte, California

Steven T. Rosen, M.D., is provost and chief scientific officer for City of Hope and a member of City of Hope’s Executive Team. He also is director of the Comprehensive Cancer Center and holds the Irell & Manella Cancer Center Director’s Distinguished Chair, and he is director of Beckman Research Institute (BRI) and the Irell & Manella Graduate School of Biological Sciences.

Dr. Rosen sets the scientific direction of City of Hope, shaping the research and educational vision for the biomedical research, treatment and education institution. Working closely and collaboratively with City of Hope’s scientists, clinicians and administrative leaders, he develops strategies that contribute to the organization’s mission.

As director of BRI, he works with faculty across the institution to help shape and direct the scientific vision for BRI while leading the vital basic and translational research that is fundamental to our strategic plan and mission. He focuses on opportunities for expanding and integrating our research initiatives; recruiting and leading talented scientists; helping our talented researchers achieve national and international recognition; and promoting our national standing as a premier scientific organization.

Prior to joining City of Hope, Dr. Rosen was the Genevieve Teuton Professor of Medicine at the Feinberg School of Medicine at Northwestern University in Chicago. He served for 24 years as director of Northwestern’s Robert H. Lurie Comprehensive Cancer Center. Under his leadership, the center received continuous National Cancer Institute (NCI) funding beginning in 1993 and built nationally recognized programs in laboratory sciences, clinical investigations, translational research and cancer prevention and control. The center attained comprehensive status in 1997.

Dr. Rosen has published more than 400 original reports, editorials, books and book chapters. His research has been funded by the National Cancer Institute, American Cancer Society, Leukemia & Lymphoma Society of America and Multiple Myeloma Research Foundation.

Dr. Rosen also has served as an adviser for several of these organizations and on the external advisory boards of more than a dozen NCI-designated Comprehensive Cancer Centers. He is the current editor-in-chief of the textbook series “Cancer Treatment & Research.”

Recognized as one of the Best Doctors in America, Dr. Rosen is a recipient of the Martin Luther King Humanitarian Award from Northwestern Memorial Hospital and the Man of Distinction Award from the Israel Cancer Research Fund. He earned his bachelor’s degree and medical degree with distinction from Northwestern University from which he also earned the Alumni Merit Award, and is a member of the Alpha Omega Alpha Honor Society.

Editor’s Note: 

We would like to thank Mary-Fran Faraji, David Caouette, and Chantal Roshetar of the Communications and Public Affairs department at the City of Hope, for the gracious help and invaluable support they provided during this interview.



The City of Hope (https://www.cityofhope.org/homepage), Duarte, California.

Other related articles

Retrieved from https://www.cityofhope.org/people/rosen-steven

Retrieved from https://www.cityofhope.org/research/beckman-research-institute

Retrieved from https://www.cityofhope.org/research/comprehensive-cancer-center

Retrieved from https://www.cityofhope.org/research/research-overview/diabetes-metabolism-research-institute

Retrieved from https://www.cityofhope.org/patients/departments-and-services/hematologic-malignancies-and-stem-cell-transplantation-institute

Retrieved from https://www.cityofhope.org/patients/departments-and-services/medical-oncology-and-therapeutics-research/medical-oncology-research

Retrieved from https://www.cityofhope.org/patients/cancers-and-treatments/departments-and-services/radiation-oncology/radiation-oncology-research


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CD-4 Therapy for Solid Tumors

Curator: Larry H. Bernstein, MD, FCAP


CD4 T-cell Immunotherapy Shows Activity in Solid Tumors

Alexander M. Castellino, PhD


For the first time, treatment with genetically engineered T-cells has used CD4 T-cells instead of the CD8 T-cells, which are used in the chimeric antigen receptor (CAR) T-cell approach. Early data suggest that this CD4 T-cell approach has activity against solid tumors, whereas the CAR T-cell approach so far has achieved dramatic success in hematologic malignancies.

In the new approach, CD4 T-cells were genetically engineered to target MAGE-A3, a protein found on many tumor cells. The treatment was found to be safe in patients with metastatic cancers, according to data from a phase 1 clinical study presented here at the American Association for Cancer Research (AACR) 2016 Annual Meeting.

“This is the first trial testing an immunotherapy using genetically engineered CD4 T-cells,” senior author Steven A. Rosenberg, MD, PhD, chief of the Surgery Branch at the National Cancer Institute (NCI), told Medscape Medical News.

Most approaches use CD8 T-cells. Although CD8 T-cells are known be cytotoxic and CD4 T-cells are normally considered helper cells, CD4 T-cells can induce tumor regression, he said.

Louis M. Weiner, MD, director of the Lombardi Comprehensive Cancer Center at Georgetown University, in Washington, DC, indicated that in contrast with CAR T-cells, these CD4 T-cells target proteins on solid tumors. “CAR T-cells are not tumor specific and do not target solid tumors,” he said.

Engineering CD4 Cells

Immunotherapy with engineered CD4 T-cells was personalized for each patient whose tumors had not responded to or had recurred following treatment with least one standard therapy. The immunotherapy was specific for patients in whom a specific human leukocyte antigen (HLA) — HLA-DPB1*0401 — was found to be expressed on their cells and whose tumors expressed MAGE-A3.

MAGE-A3 belongs to a class of proteins expressed during fetal development. The expression is lost in normal adult tissue but is reexpressed on tumor cells, explained presenter Yong-Chen William Lu, PhD, a research fellow in the Surgery Branch of the NCI.

Targeting MAGE-A3 is relevant, because it is frequently expressed in a variety of cancers, such as melanoma and urothelial, esophageal, and cervical cancers, he pointed out.

 Researchers purified CD4 T-cells from the peripheral blood of patients. Next, the CD4 T-cells were genetically engineered with a retrovirus carrying the T-cell receptor (TCR) gene that recognizes MAGE-A3. The modified cells were grown ex vivo and were transferred back into the patient.

Clinical Results

Dr Lu presented data for 14 patients enrolled into the study: eight patients received cell doses from 10 million to 30 billion cells, and six patients received up to 100 billion cells.

This was similar to a phase 1 dose-finding study, except the researchers were seeking to determine the maximum number of genetically engineered CD4 T-cells that a patient could safely receive.

One patient with metastatic cervical cancer, another with metastatic esophageal cancer, and a third with metastatic urothelial cancer experienced partial objective responses. At 15 months, the response is ongoing in the patient with cervical cancer; after 7 months of treatment, the response was durable in the patient with urothelial cancer; and a response lasting 4 months was reported for the patient with esophageal cancer.

Dr Lu said that a phase 2 trial has been initiated to study the clinical responses of this T-cell receptor therapy in different types of metastatic cancers.

In his discussion of the paper, Michel Sadelain, MD, of the Memorial Sloan Kettering Cancer Center, New York City, said, “Although therapy with CD4 cells has been evaluated using endogenous receptor, this is the first study using genetically engineered CD4 T-cells.”

Although the study showed that therapy with genetically engineered T-cells is safe and efficacious at least in three patients, the mechanism of cytotoxicity remains unclear, Dr Sadelain indicated.

Comparison With CAR T-cells

CAR T-cells act in much the same way. CARs are chimeric antigen receptors that have an antigen-recognition domain of an antibody (the V region) and a “business end,” which activates T-cells. In this case, CD8 T-cells from the patients are used to genetically engineer T-cells ex vivo. In the majority of cases, dramatic responses have been seen in hematologic malignancies.

CARs, directed against self-proteins, result in on-target, off-tumor effects, Gregory L. Beatty, MD, PhD, assistant professor of medicine at the University of Pennsylvania, in Philadelphia, indicated when he reported the first success story of CAR T-cells in a solid pancreatic cancer tumor.

Side effects of therapy with CD4 T-cells targeting MAGE-A3 were different and similar to side effects of chemotherapy, because patients received a lymphodepleting regimen of cyclophosphamide and fludabarine. Toxicities included high fever, which was experienced by the majority of patients (12/14). The fever lasted 1 to 2 weeks and was easily manageable.

High levels of the cytokine interleukin-6 (IL-6) were detected in the serum of all patients after treatment. However, the elevation in IL-6 levels was not considered to be a cytokine release syndrome, because no side effects occurred that correlated with the syndrome, Dr Liu indicated.

He also indicated that future studies are planned that will employ genetically engineered CD4 T-cells in combination with programmed cell death protein 1–blocking antibodies.

This study was funded by Intramural Research Program of the National Institutes of Health. The NCI’s research and development of T-cell receptor therapy targeting MAGE-A3 are supported in part under a cooperative research and development agreement between the NCI and Kite Pharma, Inc. Kite has an exclusive, worldwide license with the NIH for intellectual property relating to retrovirally transduced HLA-DPB1*0401 and HLA A1 T-cell receptor therapy targeting MAGE-A3 antigen. Dr Lu and Dr Rosenberg have disclosed no relevant financial relationships.

American Association for Cancer Research (AACR) 2016 Annual Meeting: Abstract CT003, presented April 17, 2016.


Searches Related to immunotherapy using genetically engineered CD4 T-cells


Genetic engineering of T cells for adoptive immunotherapy

To be effective for the treatment of cancer and infectious diseases, T cell adoptive immunotherapy requires large numbers of cells with abundant proliferative reserves and intact effector functions. We are achieving these goals using a gene therapy strategy wherein the desired characteristics are introduced into a starting cell population, primarily by high efficiency lentiviral vector-mediated transduction. Modified cells are then expanded using ex vivo expansion protocols designed to minimally alter the desired cellular phenotype. In this article, we focus on strategies to (1) dissect the signals controlling T cell proliferation; (2) render CD4 T cells resistant to HIV-1 infection; and (3) redirect CD8 T cell antigen specificity.
Adoptive T cell therapy is a form of transfusion therapy involving the infusion of large numbers of T cells with the aim of eliminating, or at least controlling, malignancies or infectious diseases. Successful applications of this technique include the infusion of CMV-or EBVspecific CTLs to protect immunosuppressed patients from these transplantation-associated diseases [1,2]. Furthermore, donor lymphocyte infusions of ex vivo-expanded allogeneic T cells have been used to successfully treat hematological malignancies in patients with relapsed disease following allogeneic hematopoietic stem cell transplant [3]. However, in many other malignancies and chronic viral infections such as HIV-1, adoptive T cell therapy has achieved inconsistent and/or marginal successes. Nevertheless, there are compelling reasons for optimism on this strategy. For example, the existence of HIV-positive elite non-progressors [4], as well as the correlation between the presence of intratumoral T cells and a favorable prognosis in malignancies such as ovarian [5,6] and colon carcinoma [7,8], provides in vivo evidence for the critical role of the immune system in controlling both HIV and cancer.
The key to successful adoptive immunotherapy strategies appears to consist of (1) using the “right” T cell type(s) and (2) obtaining therapeutically effective numbers of these cells without compromising their effector functions or their ability to engraft within the host. This article is focused on strategies employed in our laboratory to generate the “right” cell through genetic engineering approaches, with an emphasis on redirecting the antigen specificity of CD8 T cells, and rendering CD4 T cells resistant to HIV-1 infection. The article by Paulos et al. describes the evolving process of how to best obtain therapeutically effective numbers of the “right” cells by optimizing ex vivo cell expansion strategies.
Our laboratory’s overall strategy and flow plan for development and evaluation of engineered T cells is depicted in Fig. 1. We work almost exclusively with primary human T cells; little or no work is performed with conventional established cell lines. Thus, we benefit substantially from our close association with the UPenn Human Immunology Core. The Core performs leukaphereses on healthy donors 2–3 times a week, and provides purified peripheral blood mononuclear cell subsets, ensuring a constant influx of fresh human T cells into our laboratory. We have extensive experience in developing both bead- and cell-based artificial antigen presenting cells (aAPCs), as described in detail in the article by Paulos et al. The ability to genetically modify T cells at high efficiency is critical for virtually every project within the laboratory. We have adapted the lentiviral vector system described by Dull [15] for most, but not all, of the engineering applications in our laboratory.
CD4 T cells are the primary target of HIV-1, and decreasing CD4 T cell numbers is a hallmark of advancing HIV-1 disease [34]. Thus, strategies that protect CD4 T cells from HIV-1 infection in vivo would conceivably provide sufficient immunological help to control HIV-1 infection. Our early observations that CD3/CD28 costimulation resulted in improved ex vivo expansion of CD4 T cells from both healthy and HIV-infected donors, as well as enhanced resistance to HIV-1 infection [35,36], ultimately led to the first-in-human trial of lentiviral vector-modified CD4 T cells [37]. In this trial, CD4 T cells from HIV-positive subjects who had failed antiretroviral therapy were transduced with a lentiviral vector encoding an antisense RNA that targeted a 937 bp region in the HIV-1 envelope gene. Preclinical studies demonstrated that this antisense region, directed against the HIV-1NL4-3 envelope, provided robust protection from a broad range of both R5-and X4-tropic HIV-1 isolates [38]. One year after administration of a single dose of the gene-modified cells, four of the five enrolled patients had increased peripheral blood CD4 T cell counts, and in one subject, a 1.7 log decrease in viral load was observed. Finally, in two of the five patients, persistence of the gene-modified cells was detected one year post-infusion.
Since its identification as the primary co-receptor involved in HIV transmission, CCR5 has attracted considerable attention as a target for HIV therapy [42,43]. Indeed, “experiments of nature” have shown that individuals with a homozygous CCR5 Δ32 deletion are highly resistant to HIV-1 infection. Thus, we hypothesized that knocking out the CCR5 locus would generate CD4 T cells permanently resistant to infection by R5 isolates of HIV-1. To test this hypothesis we took advantage of zinc-finger nuclease (ZFN) technology [44]. ZFNs introduce sequencespecific double-strand DNA breakage, which is imperfectly repaired by non-homologous endjoining. This results in the permanent disruption of the genomic target, a process termed genome editing (Fig. 3).
Genetic modification of T cells to redirect antigen specificity is an attractive strategy compared to the lengthy process of growing T cell lines or CTL clones for adoptive transfer. Genetically modified, adoptively transferred T cells are capable of long-term persistence in humans [37, 46,47], demonstrating the feasibility of this approach. When compared to the months it can take to generate an infusion dose of antigen-specific CTL lines or clones from a patient, a homogeneous population of redirected antigen-specific cells can be expanded to therapeutically relevant numbers in about two weeks [3]. Several strategies are being explored to bypass the need to expand antigen-specific T cells for adoptive T cell therapy. The approaches currently studied in our laboratory involve the genetic transfer of chimeric antigen receptors and supraphysiologic T cell receptors.
Chimeric antigen receptors (CARs or T-bodies) are artificial T cell receptors that combine the extracellular single-chain variable fragment (scFv) of an antibody with intracellular signaling domains, such as CD3ζ or Fc(ε)RIγ [48–50]. When expressed on T cells, the receptor bypasses the need for antigen presentation on MHC since the scFv binds directly to cell surface antigens. This is an important feature, since many tumors and virus-infected cells downregulate MHCI, rendering them invisible to the adaptive immune system. The high-affinity nature of the scFv domain makes these engineered T cells highly sensitive to low antigen densities. In addition, new chimeric antigen receptors are relatively easy to produce from hybridomas. The key to this approach is the identification of antigens with high surface expression on tumor cells, but reduced or absent expression on normal tissues.  Since one can redirect both CD4 and CD8 T cells, the T-body approach to immunotherapy represents a near universal “off the shelf” method to generate large numbers of antigen-specific helper and cytotoxic T cells.
Many T-bodies targeting diverse tumors have been developed [51], and four have been evaluated clinically [52–55]. Three of the four studies were characterized by poor transgene expression and limited T-body engraftment. However, in a study of metastatic renal cell carcinoma using a T-body directed against carbonic anhydrase IX [55], T-body-expressing cells were detectable in the peripheral blood for nearly 2 months post-administration.
The major goals in the T-body field currently are to optimize their engraftment and maximize their effector functions. Our laboratory is addressing both problems simultaneously through an in-depth study of the requirements for T-body activation. We hypothesize that their limited persistence is due to incomplete cell activation due to the lack of costimulation. While naïve T cells depend on costimulation through CD28 ligation to avoid anergy and undergo full activation in response to antigen, it is recognized that effector cells also require costimulation to properly proliferate and produce cytokines [56]. Previous studies have shown that providing CD28 costimulation is crucial for the antitumoral function of adoptively transferred T cells and T-bodies [57–59]. Unlike conventional T cell activation, which requires two discrete signals, T-bodies can be engineered to provide both costimulation and CD3 signaling through one binding event.
A different approach for redirecting specificity to T cells for adoptive immunotherapy involves the genetic transfer of full-length TCR genes. A T cell’s specificity for its cognate antigen is solely determined by its TCR. Genes encoding the α and β chains of a T cell receptor (TCR) can be isolated from a T cell specific for the antigen of interest and restricted to a defined HLA allele, inserted into a vector, and then introduced into large numbers of T cells of individual patients that share the restricting HLA allele as well as the targeted antigen. In 1999, Clay and colleagues from Rosenberg’s group at the National Cancer Institute were the first to report the transfer of TCR genes via a retroviral vector into human lymphocytes and to show that T cells gained stable reactivity to MART-1 [67]. To date, many others have shown that the same approach can be used to transfer specificity for multiple viral and tumor associated antigens in mice and human systems. These T cells gain effector functions against the transferred TCR’s cognate antigen, as defined by proliferation, cytokine production, lysis of targets presenting the antigen, trafficking to tumor sites in vivo, and clearance of tumors and viral infection.
In 2006, Rosenberg’s group redirected patients’ PBLs with the naturally occurring, MART-1- specific TCR reported in 1999 by Clay. In the first clinical trial to test TCR-transfer immunotherapy, these modified T cells were infused into melanoma patients [68]. While the transduced T cells persisted in vivo, only two of the 17 patients had an objective response to this therapy. One issue revealed by the study was the poor expression of the transgenic TCRs by the transferred T cells. Nonetheless, the results from this trial showed the potential of TCR transfer immunotherapy as a safe form of therapy for cancer and highlighted the need to optimize such therapy to attain maximum potency.
The adoptive immunotherapy field is advancing by a tried-and-true method: learning from disappointments and moving forward. Our ability to fully realize the therapeutic potential of adoptive T cell therapy is tied to a more complete understanding of how human T cells receive signals, kill targets, and modulate effective immune responses. Our goal is to perform labbased experiments that provide insight into how primary T cells function in a manner that will facilitate and enable adoptive T cell therapy clinical trials. Our ability to efficiently modify (and expand) T cells ex vivo provides the opportunity to deliver sufficient immune firepower where it has heretofore been lacking. Sustained transgene expression, coupled with enhanced in vivo engraftment capability, will move adoptive immunotherapy into a realm where longterm therapeutic benefits are the norm rather than the exception.
Genetic Modification of T Lymphocytes for Adoptive Immunotherapy

Claudia Rossig1 and Malcolm K. Brenner2
Molecular Therapy (2004) 10, 5–18;   http://dx.doi.org:/10.1016/j.ymthe.2004.04.014      http://www.nature.com/mt/journal/v10/n1/full/mt20041193a.html

Adoptive transfer of T lymphocytes is a promising therapy for malignancies—particularly of the hemopoietic system—and for otherwise intractable viral diseases. Efforts to broaden the approach have been limited by the physiology of the T cells themselves and by a range of immune evasion mechanisms developed by tumor cells. In this review we show how genetic modification of T cells is being used preclinically and in patients to overcome these limitations, by incorporation of novel receptors, resistance mechanisms, and control genes. We also discuss how the increasing safety and effectiveness of gene transfer technologies will lead to an increase in the use of gene-modified T cells for the treatment of a wider range of disorders.

That gene transfer could be used to improve the effectiveness of T lymphocytes was apparent from the beginning of clinical studies in the field. T cells were the very first targets for genetic modification in human gene transfer experiments. Rosenberg’s group marked tumor-infiltrating lymphocytes ex vivo with a Moloney retroviral vector encoding neomycin phosphotransferase before reinfusing them and attempting to demonstrate selective accumulation at tumor sites. Shortly thereafter, Blaese and Anderson led a group that infused corrected T cells into two children with severe combined immunodeficiency due to ADA deficiency. While neither study was completely successful in terms of outcome, both showed the feasibility of ex vivo gene transfer into human cells and set the stage for many of the studies that followed. More recently, a second wave of interest in adoptive T cell therapies has developed, based on their success in the prevention and treatment of viral infections such as EBV and cytomegalovirus (CMV) and on their apparent ability to eradicate hematologic and perhaps solid malignancies1,2,3,4,5,6. There has been a corresponding increase in studies directed toward enhancing the antineoplastic and antiviral properties of the T cells. In this article we will review how gene transfer may be used to produce the desired improvements focusing on vectors and genes that have had clinical application.

Currently available viral and nonviral vector systems lack a pattern of biodistribution that would favor T cell transduction in vivo—as occurs, for example, with adenovectors and the liver or liposomal vectors and the lung. This lack of favorable biodistribution cannot yet be compensated for by the introduction of specific T-cell-targeting ligands into vectors. Hence, all T cell gene transfer studies conducted to date have used ex vivo transduction followed by adoptive transfer of gene-modified cells. This approach is inherently less attractive for commercial development than directin vivo gene transfer and has probably restricted interest in developing clinical applications using these cells. On the other hand, ex vivo transduction may be more readily controlled, characterized, and standardized than in vivo efforts and may ultimately produce a better defined final product (the transduced cell).

The gene products of suicide and coexpressed resistance genes are highly immunogenic and may induce immune-mediated rejection of the transduced cells. In one study, the persistence of adoptively transferred autologous CD8+ HIV-specific CTL clones modified to express the hygromycin phosphotransferase (Hy) gene and the herpesvirus thymidine kinase gene as a fusion gene was limited by the induction of a potent CD8+ class I MHC-restricted CTL response specific for epitopes derived from the Hy-tk protein126. Less immunogenic suicide and selection marker genes, preferably of human origin, may reduce the immunological inactivation of genetically modified donor lymphocytes. Human-derived prodrug-activating systems include the human folylpolyglutamate synthetase/methotrexate127, the deoxycytidine/cytosine arabinoside128, or the carboxylesterase/irinotecan129 systems. These systems do not activate nontoxic prodrugs but are based on enhancement of already potent chemotherapeutic agents. The administration of methotrexate to treat severe GVHD may not only kill transduced donor lymphocytes but may also have additional inhibitory activity on nontransduced but activated T cells.

Finally, endogenous proapoptotic molecules have been proposed as nonimmunogenic suicide genes. A chimeric protein that contains the FK506-binding protein FKBP12 linked to the intracellular domain of human Fas130 was recently introduced. Addition of the dimerizing prodrug induces Fas crosslinking with subsequent triggering of an apoptotic death signal.

Genetic engineering of T lymphocytes should help deliver on the promise of immunotherapies for cancer, infection, and autoimmune disease. Improvements in transduction, selection, and expansion techniques and the development of new viral vectors incapable of insertional mutagenesis will reduce the risks and further enhance the integration of T cell and gene therapies. Nonetheless, successful application of the proposed modifications to the clinical setting still requires many iterative studies to allow investigators to optimize the individual components of the approach.

Genetically modified T cells in cancer therapy: opportunities and challenges
Michaela Sharpe, Natalie Mount


The feasibility of T-cell adoptive transfer was first reported nearly 20 years ago (Walter et al., 1995) and the field of T-cell therapies is now poised for significant clinical advances. Recent clinical trial successes have been achieved through multiple small advances, improved understanding of immunology and emerging technologies. As the key challenges of T-cell avidity, persistence and ability to exert the desired anti-tumour effects as well as the identification of new target antigens are addressed, a broader clinical application of these therapies could be achieved. As the clinical data emerges, the challenge of making these therapies available to patients shifts to implementing robust, scalable and cost-effective manufacture and to the further evolution of the regulatory requirements to ensure an appropriate but proportionate system that is adapted to the characteristics of these innovative new medicines.



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A Mechanism of Cancer Metastasis

Larry H. Bernstein, MD, FCAP, Curator


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Word Cloud By Danielle Smolyar

A Protein that Spreads Cancer

Nils Halberg at the University of Bergen has identified a protein that makes it possible for cancer cells to spread


The cells inside a tumour differ a lot. While some remains “good” and do not cause trouble, others become aggressive and starts to spread to other organ sites. It is very hard to predict which cells become aggressive or not.

Nevertheless, by isolating these aggressive cancer cells in in vivotests on animals, Nils Halberg at the Department of Biomedicinet the University of Bergen (UiB) and the researchers Dr. Sohail Tavazoie and Dr. Caitlin Sengelaub at The Rockefeller University have discovered a certain protein (PITPNC1) that characterise aggressive cancer cells.

“We discovered that the aggressive cancer cells that are spreading in colon, breast, and skin cancer contained a much higher portion of the protein PITPNC1, than the non-aggressive cancer cells,” says researcher Nils Halberg of the CELLNET Group at the Department of Biomedicine at UiB.

“This means we can predict which of the cancer cells are getting aggressive and spread, at a much earlier stage than today.”

How cells penetrate tissue

The researcher also discovered that this protein, that characterizes the aggressive cancer cells, has got a very specific function in the process of spreading cancer.

The cancer cells spread from one place in the body to another, through the blood vessel. To get into the blood vessels, the cell needs to penetrate tissue, both when it leaves the tumour and when it is attaching to a new organ.

“The protein PITPNC1 regulates a process whereby the cancer cells are secreting molecules, which cut through a network of proteins outside the cells, like scissors. The cancer cell is then able to penetrate the tissue and set up a colonies at new organ sites,” Halberg explains.

Custom-made therapy

A tumour that is not spreading, is usually not dangerous for the patient if it is removed. The hard part in cancer therapy is when the tumour starts to spread. Guided by the new discoveries, supported by the Bergen Research Foundation´s (BFS) Recruitment Programme, Halberg hopes to contribute to a better treatment of cancer patients.

“If we get to the point where we can offer a custom-made therapy that targets the function of this protein, we might be able to stop it spreading,” says Nils Halberg.


This gene encodes a member of the phosphatidylinositol transfer protein family. The encoded cytoplasmic protein plays a role in multiple processes including cell signaling and lipid metabolism by facilitating the transfer of phosphatidylinositol between membrane compartments. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene, and a pseudogene of this gene is located on the long arm of chromosome 1. [provided by RefSeq, May 2012]

Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid*

Kathryn Garner‡ , Alan N. Hunt§ , Grielof Koster§ , Pentti Somerharju¶ , Emily Groves‡1, Michelle Li‡ , Padinjat Raghu , Roman Holic‡ , and Shamshad Cockcroft‡2
JBC Papers in Press, July 21, 2012,      http://dx.doi.org:/10.1074/jbc.M112.375840

Background: Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgB) promotes metastatic colonization and angiogenesis in humans.

Results: We demonstrate that RdgB is a phosphatidic acid (PA)- and phosphatidylinositol-binding protein and binds PA derived from the phospholipase D pathway.

Conclusion: RdgB is the first lipid-binding protein identified that can bind and transfer PA.

Significance: PA bound to RdgB is a likely effector downstream of phospholipase D

PITPNC1 Recruits RAB1B to the Golgi Network to Drive Malignant Secretion

Nils Halberg3,4,, Caitlin A. Sengelaub3, Kristina Navrazhina, Henrik Molina, Kunihiro Uryu, Sohail F. Tavazoie
Cancer Cell 14 Mar 2016; Volume 29, Issue 3:339–353   http://dx.doi.org/10.1016/j.ccell.2016.02.013
  • PITPNC1 promotes metastasis by melanoma, breast cancer, and colon cancer cells
  • PITPNC1 recruits RAB1B to the Golgi compartment of the cell
  • Golgi localization of RAB1B enhances vesicular secretion via GOLPH3 recruitment


Enhanced secretion of tumorigenic effector proteins is a feature of malignant cells. The molecular mechanisms underlying this feature are poorly defined. We identify PITPNC1 as a gene amplified in a large fraction of human breast cancer and overexpressed in metastatic breast, melanoma, and colon cancers. Biochemical, molecular, and cell-biological studies reveal that PITPNC1 promotes malignant secretion by binding Golgi-resident PI4P and localizing RAB1B to the Golgi. RAB1B localization to the Golgi allows for the recruitment of GOLPH3, which facilitates Golgi extension and enhanced vesicular release. PITPNC1-mediated vesicular release drives metastasis by increasing the secretion of pro-invasive and pro-angiogenic mediators HTRA1, MMP1, FAM3C, PDGFA, and ADAM10. We establish PITPNC1 as a PI4P-binding protein that enhances vesicular secretion capacity in malignancy.

Cancerous Conduits

Metastatic cancer cells use nanotubes to manipulate blood vessels.

By Amanda B. Keener | April 1, 2016



MAKING CONTACT: Breast cancer cells (white arrows) in culture deliver microRNAs to endothelial cells through filamentous nanotubes (yellow arrow).


The paper
Y. Conner et al., “Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype,” Nat Commun, 6:8671, 2015.

Branching Out
Harvard bioengineer Shiladitya Sengupta and his team were establishing a culture system to model the matrix and blood vessel networks that surround tumors when they found that human breast cancer cells spread out along blood vessel endothelial cells rather than form spheroid tumors as expected. Taking a closer look using scanning electron microscopy, they spied nanoscale filaments consisting of membrane and cytoskeletal components linking the two cell types.

Manipulative Metastases
These cancer cell–spawned nanotubes, the team discovered, could transfer a dye from cancer cells to endothelial cells both in culture and in a mouse model of breast cancer metastasis to the lungs.The cells also transferred microRNAs known to regulate endothelial cell adhesion and disassociation of tight junctions, which Sengupta speculates may help cancer cells slip in and out of blood vessels. This study is the first to suggest a role for nanotubes in metastasis.

Breaking the Chain
Sengupta’s team then used low doses of cytoskeleton-disrupting drugs to block nanotube formation. Emil Lou, an oncologist at the University of Minnesota who studies nanotubes in cancer and was not involved in the study, says this approach is a “good start,” though such drugs would not be used in human patients because they are not specific to nanotubes.

In the Details
Lou says the study emphasizes the importance of understanding interactions between tumors and their surrounding tissues on a molecular level. Going forward, Sengupta plans to study how the tubes are formed in melanoma as well as breast and ovarian cancers to try to identify other drug targets.

Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype

Yamicia ConnorSarah TekleabShyama NandakumarCherelle Walls,….., Bruce ZetterElazer R. Edelman & Shiladitya Sengupta
Nature Communications6,Article number:8671

Metastasis is a major cause of mortality and remains a hurdle in the search for a cure for cancer. Not much is known about metastatic cancer cells and endothelial cross-talk, which occurs at multiple stages during metastasis. Here we report a dynamic regulation of the endothelium by cancer cells through the formation of nanoscale intercellular membrane bridges, which act as physical conduits for transfer of microRNAs. The communication between the tumour cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits. These results lead us to define the notion of ‘metastatic hijack’: cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits. Pharmacological perturbation of these nanoscale membrane bridges decreases metastatic foci in vivo. Targeting these nanoscale membrane bridges may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer.

Metastasis is the culmination of a cascade of events, including invasion and intravasation of tumour cells, survival in circulation, extravasation and metastatic colonization4. Multiple studies have reported a dynamic interaction between the metastatic tumour cell and the target organ, mediated by cytokines4, 12 or by exosomes that can prime metastasis by creating a pre-metastatic niche13. Interestingly, the interactions between cancer cells and endothelium in the context of metastasis, which occurs during intravasation, circulation and extravasation, remains less studied. Cancer cell-secreted soluble factors can induce retraction of endothelial cells and the subsequent attachment and transmigration of tumour cells through the endothelial monolayers14, 15. Recently, studies indicate a more intricate communication between cancer cells and the endothelium. For example, a miRNA regulon was found to mediate endothelial recruitment and metastasis by cancer cells16. Similarly, exosome-mediated transfer of cancer-secreted miR-105 was recently reported to disrupt the endothelial barrier and promote metastasis17. We rationalized that a better understanding of cancer–endothelial intercellular communication, primarily during extravasation, could lead to novel strategies for inhibiting metastasis18.

Recently, nanoscale membrane bridges, such as tunnelling nanotubes (TNTs) and filopodias, have emerged as a novel mechanism of intercellular communication19. For example, specialized signalling filopodia or cytonemes were recently shown to transport morphogens during development20. Similarly, TNTs, which unlike filopodia have no contact with the substratum21, were shown to facilitate HIV-1 transmission between T cells, enable the spread of calcium-mediated signal between cells and transfer p-glycoproteins conferring multi-drug resistance between cancer cells22, 23, 24, 25. TNTs were also recently implicated in trafficking of mitochondria from endothelial to cancer cells and transfer miRNA between osteosarcoma cells and stromal murine osteoblast cells, and between smooth muscle cells and the endothelium26, 27, 28. However, whether similar intercellular nanostructure-mediated communication can be harnessed by cancer cells to modulate the endothelium is not known.

Here we report that metastatic cancer cells preferentially form nanoscale intercellular membrane bridges with endothelial cells. These nanoscale bridges act as physical conduits through which the cancer cells can horizontally transfer miRNA to the endothelium. We observe that the recipient endothelial cells present an miRNA profile that is distinct from non-recipient endothelial cells isolated from the same microenvironment. Furthermore, the co-cultures of cancer and endothelial cells upregulate markers associated with pathological endothelium, which is inhibited by pharmacological disruption of the nanoscale conduits. Additionally, the pharmacological inhibitors of these nanoscale conduits can decrease metastatic foci in vivo, which suggests that these nanoscale conduits may potentially emerge as new targets in the management of metastatic cancer.

Figure 1: Nanoscale structures physically connect metastatic cells and the endothelium

Nanoscale structures physically connect metastatic cells and the endothelium.


(a) Representative image of MDA-MB-231 cancer cells exhibiting an invasive phenotype in the presence of preformed endothelial tubes in co-culture. (b) Representative image of a mammosphere typically formed by MDA-MB-231 cells when cultured on 3D tumour matrix in the absence of endothelial cells. MDA-MB-231 cells were loaded with CFSE. Actin was labelled with rhodamine phalloidin and nuclei were counterstained with DAPI. (c) A representative SEM of epithelial (EPI) MDA-MB-231 cells aligning on HUVEC (ENDO) tubules in the co-culture. Lower panel shows higher magnification. (d) SEM image reveals nanoscale membrane bridges connecting (nCs) metastatic breast cancer (EPI) cells and endothelial vessels (arrows). (e) A representative transmission electron micrograph shows intercellular connectivity through the nanoscale membrane bridge between MDA-MB-231 and an endothelial cell. (f) A cartoon represents the types of homotypic and heterotypic intercellular nanoscale connections that an epithelial cell may form in the presence of endothelial tubules. Highly metastatic (MDA-MB-468, MDA-MB-231 or MDA-MB-435) or low metastatic (MCF7 and SkBr3) cancer cells were co-cultured with the endothelial tubes. Normal HMECs were used as control. Graphs show percentage of total population of epithelial cells that exhibit either homotyptic (Epi–Epi) or heterotypic (Epi–Endo) nanoscale connections and (g) average number of nanoscale connections formed per cell. Quantification analysis was done on >300 cells of each cell type. Data shown are mean±s.e.m. (n=6 replicates per study, with 2–3 independent experiments). **P<0.01, ***P<0.001 (analysis of variance followed by Bonferroni’s post-hoctest).

The nanoscale bridges are composed of cytoskeletal elements

Figure 3: Structure and function of the heterotypic intercellular nanoscale membrane bridges.

a) Representative images show the heterotypic nanoscale membrane bridges are composed of both F-actin and α/β-tubulin cytoskeletal components. Co-cultures were stained with α/β-tubulin antibody (green) and phalloidin (purple) to label actin, and counterstained with DAPI (nuclear)+WGA (plasma membrane) (blue). Endothelial cells were labelled with DiL-Ac-LDL (red). (b) Mathematical modelling of the structure of the nanoscale connections. The physical properties of actin filaments necessitate microtubules for projections of certain length scales. The maximum projection length for a given minimum diameter at the buckling limit is plotted for actin-only nanoscale structures (purple line). This curve is overlaid with the experimental length and diameter measurements (red dots) from the observed thin projections measured in these studies. Projections containing only actin or projections containing both actin and tubulin can exist to the right of the curve (purple line). However, actin-only projections cannot exist to the left of the curve (green region). (c) The effect of incorporating tubulin in these projections. The maximum length is plotted against the minimum diameter for varying fractions of tubulin incorporated in the nanoscale projection. Addition of microtubules to the projections increases the overall flexural rigidity, shifting the curves left of the actin-only limit (purple line), thus allowing for longer and thinner nanoscale connections. However, owing to the larger radius of microtubules (4 × radius of actin filaments), there is an optimal fraction of tubulin (green line) that can be incorporated into the projection before the effect is reversed. (d) The optimal fraction of microtubules is about 6.6% (red dashed line) to maximize nanostructure flexural strength, while minimizing thickness. (e) Representative confocal image shows the presence of myosin V motor proteins within the intercellular nanostructure (inset shows higher magnification). Scale bar 10μm.

Nanoscale bridges act as conduits for communication

Figure 4: The nanoscale membrane bridges act as conduits for intercellular communication between cancer and endothelial cells.

(a) Confocal image of nanoscale membrane bridge-mediated transfer of cytoplasmic contents. CFSE (green)-loaded MDA-MB-231 cells were co-cultured with the Dil-Ac-LDL (red)-labelled HUVECs. Transfer of the CFSE dye was observed after 24-h co-culture. CFSE dye can be seen within HUVEC cells (yellow arrow). Tumour cells can form a nanobridge with a distal endothelial cell (EC1) than an endothelial cell (EC2) in close proximity. (b,c) Cartoon shows the experimental design, where dual cultures control for vesicle-mediated intercellular transfer. FACS plot show gating for sorting endothelial cells from the co-cultures using dual staining for DiI-Ac-LDL and PECAM-1, and then quantification for CFSE transfer in the isolated endothelial cells. (d) Graph shows quantification of FACS analysis, highlighting increased transfer of CFSE to endothelial cells in the co-culture. (N>100,000 events, n=36 replicates, 3 replicates per study). (e) Graph shows the temporal kinetics of nanoscale connection-mediated intercellular transfer of CFSE from MDA-MB-231 cells to the endothelium (n=2 studies, 3 replicates per study). (f) Effect of small molecule inhibitors of cytoskeletal components on membrane nanobridges. (g) Graphs show treatment with vehicle (control) or a low-dose combination of docetaxel and cytochalasin do not affect the exosome shedding (n=2 independent studies). (h,i) Graphs show the effect of pharmacological inhibitors on the formation of heterotypic and homotypic nanoscale bridges (arrows). (n=2 studies, 6 replicates per study). (j) Graph shows the effect of pharmacological inhibitors on intercellular transfer of CFSE to endothelial cells from cancer cells (n=10 studies, 3 replicates per study). Data shown are mean±s.e.m. (*P<0.05,**P<0.01, ****P<0.001, analysis of variance followed by Bonferroni’s post-hoc test).

Effect of pharmacological inhibition of nanoscale bridges

Nanobridges transfer miRNA from cancer cells to endothelium

Figure 5: The nanoscale membrane bridges act as conduits for intercellular transfer of miRNA between cancer and endothelial cells.

Representative confocal images show the transfer of Cy3-labelled miRNA from MDA-MB-231 cells (EPI) to endothelial cells (ENDO) at (a) 24h and (b) 36h of co-culture. Alexa Fluor 488-Ac-LDL (green)-labelled endothelial cells were co-cultured with Cy3-labelled miRNA-transfected MDA-MB-231. Co-cultures were counterstained with phalloidin (purple) and DAPI+WGA (blue). A 3D visualization shows the localization of miRNA within the nanoscale connections (white arrows), which act as conduits for horizontal transfer of miRNAs to endothelial cells. (c) Schema shows quantification of Cy3-labelled control miRNA and Cy3-labelled miR132 transfer between cancer cell and endothelium using flow cytometry. Endothelial cell populations were isolated from the co-cultures and percentage of miRNA+ve cells was determined. Dual cultures in Boyden chambers were included as controls. (d) Graph shows the effect of pharmacological disruption of nanoscale conduits on miRNA transfer. (e) Schema shows experimental design for reverse transcriptase–PCR-based detection of transferred miR-132 in endothelial cells under different experimental conditions. MDA-MB-231 cells transfected with miR-132 and α-miR-132 were co-cultured with endothelial tubes. FACS-isolated endothelial cell populations were analysed for the expression of miR-132. (f) Graph shows miR-132+ve cell populations (solid red) show 5 × increase compared with miR-132−ve populations (solid blue) (P<0.0001), whereas anti-miR-132+ve cells (striped red) show 26 × decrease in miR-132 expression (P<0.0001) compared with α-miR-132−ve cells (striped blue). Direct transfection of miR-132 (black) and α-miR-132 (light blue) in endothelial cells is used as positive and negative controls, respectively. Upregulation of miR-132 from baseline was observed in dual culture (solid green), which could be inhibited with anti-miR-132 (striped green). MiR-132 levels are increased compared with dual only in those cells that are positive for intercellular transfer. Fold change was determined compared with endothelial cell transfection with control miRNA (grey). (g) FACS analysis shows nanoscale bridges-mediated transfer of miRNAs leads to changes in p120RasGAP and pAkt (S473) expression downstream of the miR-132 pathway in endothelial cell populations isolated from co-cultures. (h) Graphs show p120RasGAP expression is decreased in the miR-132+ve cell populations and increased in the α-miR-132+ve cell populations, while further downstream miR-132 positively regulates pAkt expression. Data shown are mean±s.e.m. (N=2–5 independent studies, with 3 replicates per study,*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, analysis of variance followed by Bonferroni’s post-hoctest).

Nanobridge-mediated transfer alters endogenous miRNA profile

Figure 6: Cancer cell–endothelial intercellular transfer alters the endogenous miRNA profile and phenotype of recipient endothelial cells.

The complexity of regulatory tumour parenchyma–endothelial communication is increasingly being unravelled7, 50. The altered phenotypic behaviour of the metastatic cancer cells in the presence of endothelial cells observed in this study, instead of forming classical mammospheres, is consistent with the emerging paradigm of modulatory tumour parenchyma–stroma communication and the creation of a pre-metastatic niche. Indeed, a recent study proposed the concept of the formation of a pre-metastatic niche mediated via metastatic cell-secreted exosomes, leading to vascular leakiness at the pre-metastatic sites13. Here we demonstrate that cancer cells form nanoscale membrane bridges, which can act as conduits for horizontal transfer of miRNA from the cancer cells to the endothelium, switching the latter to a pathological phenotype. Our findings reveal that the ability to form the nanoscale conduits with endothelial cells correlates with the metastatic potential of the cancer cell, and that the pharmacological perturbation of these nanoscale connections can lead to a reduction in the metastatic burden in experimental metastasis models. Together, our studies shed new insights into the tumour parenchyma–endothelial communication, adding depth to the emerging paradigm of the ability of a cancer cell to ‘hijack’ a physiological stromal cell for self-gain13.

Indeed, exosomes have emerged as an extensively studied mechanism of horizontal intercellular transfer of information51. However, a key distinction exists between the exosome-mediated versus the nanoscale membrane bridge-mediated intercellular communication. Although the former is stochastic, that is, it is unlikely the cancer cell has control over which cell will be targeted by a secreted exosome, the communication via nanoscale membrane bridges is deterministic, that is, the cancer cell can connect to a specific endothelial cell, which could be further away than the most proximal endothelial cell.

Although the aim of this study was to study the nanoscale membrane bridges as a mode of horizontal transfer of miRNAs from the metastatic cancer cells to the endothelium, and not to characterize a specific miRNA that are implicated in metastasis, many of the miRNAs, which were differentially regulated in the recipient endothelial cells, have previously been shown to regulate metastasis (Supplementary Discussion).

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Tumor Shrinking Triple Helices

Larry H. Bernstein, MD, FCAP, Curator



Tumor-Shrinking Triple-Helices

A braided structure and some adhesive hydrogel make therapeutic microRNAs both stable and sticky.

By Ruth Williams | April 1, 2016



MicroRNAs (miRs) are small, noncoding ribonucleic acids that control the translation of target messenger RNAs (mRNAs). Given their roles in development, differentiation, and other cellular processes, misregulation of miRs can contribute to diseases such as cancer. Indeed, “they are recognized as important modulators of cancer progression,” says Natalie Artzi of Harvard Medical School.

In addition to occasionally promoting cancer pathology, miRs also hold the potential to treat it—either by restoring levels of suppressed miRs, or by repressing overactive ones using antisense miRs (antagomiRs). While miRs are promising therapeutic molecules, says Daniel Siegwart of the University of Texas Southwestern Medical Center in Dallas, their use “is currently hindered by at least two issues: nucleic acid instability in vivo, and the development of effective delivery systems to transport miRs into tumor cells.”

Artzi and her team have now addressed both of these issues in one fell swoop. They first assembled two therapeutic miRs—one antagomiR and one that replaced a deficient miR—together with a third miR, a complement of the replacement strand, into triple-helix structures, which increased molecular stability without affecting function. They then complexed these helices with dendrimers—large synthetic branching polymer particles—and mixed these complexes with dextran aldehyde to form an adhesive hydrogel. The gel could then be applied directly to the surface of tumors to deliver the therapeutic miRs into cells with high efficiency.

In mice with induced breast tumors, the triple-helix–hydrogel approach led to dramatic tumor shrinkage and extended life span: the animals survived approximately one month longer than those treated with standard-of-care chemotherapy drugs. Because the RNA-hydrogel mixture must be applied directly to the tumor, the approach will not be suitable for all cancers. But one potential application, says Siegwart, is that “the hydrogel could be applied by a surgeon after performing bulk tumor removal…[and] might kill remaining tumor cells that would otherwise cause tumor recurrence.” (Nature Materials, http://dx.doi.org:/10.1038/NMAT4497, 2015)

STICKING IT TO TUMORS: To deliver therapeutic microRNAs (miRs) to tumors, braids of three microRNAs (miRs)—an antisense strand that blocks a miR overactive in cancer, a strand that replaces a deficient miR, and a stabilizing strand (1)—are added to a dendrimer (2) and mixed with a hydrogel scaffold (3). When researchers introduced the sticky gel onto mouse mammary tumors (4), the malignancies shrank and the animals lived longer (5)© GEORGE RETSECK; J.CONDE ET AL., NATURE MATERIALS


Nanoparticles Examples: gold particles, liposomes, peptide nucleic acids, or polymers Usually multiple injections Combining miRs with aptamers or antibodies can guide nanoparticles to target cells, but systemic delivery inevitably leads to some off-target dispersion. Multisite or blood cancers
RNA–triple-helix-hydrogel Dendrimer-dextran hydrogel One Adhesive hydrogel sticks miRs to tumor site with minimal dispersion to other tissues. Solid Tumors


Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment

João CondeNuria OlivaMariana AtilanoHyun Seok Song & Natalie Artzi
Nature Materials15,353–363(2016)

The therapeutic potential of miRNA (miR) in cancer is limited by the lack of efficient delivery vehicles. Here, we show that a self-assembled dual-colour RNA-triple-helix structure comprising two miRNAs—a miR mimic (tumour suppressor miRNA) and an antagomiR (oncomiR inhibitor)—provides outstanding capability to synergistically abrogate tumours. Conjugation of RNA triple helices to dendrimers allows the formation of stable triplex nanoparticles, which form an RNA-triple-helix adhesive scaffold upon interaction with dextran aldehyde, the latter able to chemically interact and adhere to natural tissue amines in the tumour. We also show that the self-assembled RNA-triple-helix conjugates remain functional in vitro and in vivo, and that they lead to nearly 90% levels of tumour shrinkage two weeks post-gel implantation in a triple-negative breast cancer mouse model. Our findings suggest that the RNA-triple-helix hydrogels can be used as an efficient anticancer platform to locally modulate the expression of endogenous miRs in cancer.


Figure 1: Self-assembled RNA-triple-helix hydrogel nanoconjugates and scaffold for microRNA delivery.

Self-assembled RNA-triple-helix hydrogel nanoconjugates and scaffold for microRNA delivery.

a, Schematic showing the self-assembly process of three RNA strands to form a dual-colour RNA triple helix. The RNA triplex nanoparticles consist of stable two-pair FRET donor/quencher RNA oligonucleotides used for in vivo miRNA inhibit…


Figure 4: Proliferation, migration and survival of cancer cells as a function of RNA-triple-helix nanoparticles treatment.close

Proliferation, migration and survival of cancer cells as a function of RNA-triple-helix nanoparticles treatment.

a, miR-205 and miR-221 expression in breast cancer cells at 24, 48 and 72h of incubation (n = 3, statistical analysis performed with a two-tailed Students t-test, , P < 0.01). miRNA levels were normalized to the RNU6B reference gene


  1. Kasinski, A. L. & Slack, F. J. MicroRNAs en route to the clinic: Progress in validating and targeting microRNAs for cancer therapy. Nature Rev. Cancer 11, 849864 (2011).
  2. Li, Z. & Rana, T. M. Therapeutic targeting of microRNAs: Current status and future challenges. Nature Rev. Drug Discov. 13, 622638 (2014).
  3. Yin, H. et al. Non-viral vectors for gene-based therapy. Nature Rev. Genet. 15, 541555(2014).
  4. Conde, J., Edelman, E. R. & Artzi, N. Target-responsive DNA/RNA nanomaterials for microRNA sensing and inhibition: The jack-of-all-trades in cancer nanotheranostics? Adv. Drug Deliv. Rev. 81, 169183 (2015).
  5. Chen, Y. C., Gao, D. Y. & Huang, L. In vivo delivery of miRNAs for cancer therapy: Challenges and strategies. Adv. Drug Deliv. Rev. 81, 128141 (2015).
  6. Yin, P. T., Shah, B. P. & Lee, K. B. Combined magnetic nanoparticle-based microRNA and hyperthermia therapy to enhance apoptosis in brain cancer cells. Small 10, 41064112(2014).
  7. Hao, L. L., Patel, P. C., Alhasan, A. H., Giljohann, D. A. & Mirkin, C. A. Nucleic acid–gold nanoparticle conjugates as mimics of microRNA. Small 7, 31583162 (2011).
  8. Endo-Takahashi, Y. et al. Systemic delivery of miR-126 by miRNA-loaded bubble liposomes for the treatment of hindlimb ischemia. Sci. Rep. 4, 3883 (2014).
  9. Chen, Y. C., Zhu, X. D., Zhang, X. J., Liu, B. & Huang, L. Nanoparticles modified with tumor-targeting scFv deliver siRNA and miRNA for cancer therapy. Mol. Ther. 18, 16501656(2010).
  10. Anand, S. et al. MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis. Nature Med. 16, 909914 (2010).


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Cambridge Healthtech Institute’s Third Annual

Clinical NGS Assays

Addressing Validation, Standards, and Clinical Relevance for Improved Outcomes

August 23-24, 2016 | Grand Hyatt Hotel | Washington, DC

Reporter: Stephen J. Williams, PhD

View Preliminary Agenda

Molecular diagnostics, particularly next-generation sequencing (NGS), have become an integral component of disease diagnosis. Still, there is work to be done to establish these tools as the standard of care. The Third Annual Clinical NGS Assays event will address NGS assay validation, establishing NGS standards, and determining clinical relevance. The pros and cons of various techniques such as gene panels, whole exome, and whole genome sequencing will also be debated with regards to depth of coverage, clinical utility, and reimbursement. Overall, this event will address the needs of both researchers and clinicians while exploring strategies to increase collaboration for improved patient outcomes.

Special Early Registration Savings Available
Register Now to Save up to $450

Preliminary Agenda


Best Practices for Using Genome in a Bottle Reference Materials to Benchmark Variant Calls
Justin Zook, National Institute of Standards and Technology

NGS in Clinical Diagnosis: Aspects of Quality Management
Pinar Bayrak-Toydemir, M.D., Ph.D., FACMG, Associate Professor, Pathology, University of Utah; Medical Director, Molecular Genetics and Genomics, ARUP Laboratories

Thorough Validation and Implementation of Preimplantation Genetic Screening for Aneuploidy by NGS
Rebekah Zimmerman, Ph.D., Laboratory Director, Clinical Genetics, Foundation for Embryonic Competence


Are We There Yet? The Odyssey of Exome Analysis and Interpretation
Avni B. Santani, Ph.D., Director, Genomic Diagnostics, Pathology and Lab Medicine, The Children’s Hospital of Philadelphia

Challenges in Exome Interpretation: Intronic Variants
Rong Mao, M.D., Associate Professor, Pathology, University of Utah; Medical Director, Molecular Genetics and Genomics, ARUP Laboratories

Exome Sequencing: Case Studies of Diagnostic and Ethical Challenges
Lora J. H. Bean, Ph.D., Assistant Professor, Human Genetics, Emory University


Implementing Analytical and Process Standards
Karl V. Voelkerding, M.D., Professor, Pathology, University of Utah; Medical Director for Genomics and Bioinformatics, ARUP Laboratories

Assuring the Quality of Next-Generation Sequencing in Clinical Laboratory Practice
Shashikant Kulkarni, M.S., Ph.D., Professor, Pathology and Immunology; Head of Clinical Genomics, Genomics and Pathology Services; Director, Cytogenomics and Molecular Pathology, Washington University at St. Louis

Sponsored Presentation to be Announced by Genection


John Chiang, Ph.D., Director, Casey Eye Institute, Oregon Health & Science University
Avni B. Santani, Ph.D., Director, Genomic Diagnostics, Pathology and Lab Medicine, The Children’s Hospital of Philadelphia
Additional Panelist to be Announced


Utility of Implementing Clinical NGS Assays as Standard-of-Care in Oncology
Helen Fernandes, Ph.D., Pathology & Laboratory Medicine, Weill Cornell Medical College

An NGS Inter-Laboratory Study to Assess Performance and QC – Sponsored by Seracare
Andrea Ferreira-Gonzalez, Ph.D., Chair, Molecular Diagnostics Division, Pathology, Virginia Commonwealth University Medical School

This conference is part of the Eighth Annual Next-Generation Dx Summit.

Track Sponsor: SeraCare

For exhibit & sponsorship opportunities, please contact:

Joseph Vacca, M.Sc.
Associate Director, Business Development
Cambridge Healthtech Institute
T: (+1) 781-972-5431
E: jvacca@healthtech.com

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Compassionate Care

Larry H. Bernstein, MD, FCAP, Curator



What Price Compassion?



“When a person realizes he has been deeply heard, his eyes moisten. I think in some real sense he is weeping for joy. It is as though he were saying, ‘Thank God, somebody heard me. Someone knows what it’s like to be me.’”

“If I let myself really understand another person, I might be changed by that understanding. And we all fear change. So as I say, it is not an easy thing to permit oneself to understand an individual.”

— Carl Rogers, American Psychologist (1902–1987)

Oncologists, whether they like it or not, must develop some psychological skills if they ever hope to master the art of caring for people living with cancer. Among our many duties we serve as therapists to those diagnosed with, living with, and dying with cancer. Therefore, it behooves us to recognize the benefits of communicating our regard for our patients’ lives and our concern for their anxieties. Compassion, defined as sympathy for another’s woes and a desire to ease them, is succor for fear. Compassion creates a bond of trust between doctors and patients that soothes painful emotions and provides support during difficult times. Given the oncologist’s busy schedule, is compassion a superficial gratuity or does it require training and execution in order to be meaningful? How do we, who have no formal training as therapists, learn to value it for our patients and use it successfully?

The eminent psychologist Carl Rogers, known as the father of client-centered therapy and the author of the two quotations above, would be a welcome addition to the oncology staff. His philosophy of therapy emphasized letting the client (his term for patients) direct the course of discussion as a means toward deeper understanding, and he emphasized the need for the therapist to follow certain guidelines. I believe his method fits perfectly with our need to learn the skill of compassion. Let’s look at the three qualities Rogers requires the therapist to possess and how they can be used in the oncology clinic.

1. Congruence, also known as Genuineness. This is the ability to be real, to be transparent, with no façade of self-importance or didactic formality that could build a wall between the patients and us. In order to express compassion to the needy, we must project an honest image of ourselves; we must drop the mask hiding our true feelings. For example, if I’m having a bad day, I should admit it rather than act frustrated for no reason. If something funny comes to me, I will share it. I want to let my patients see me for who I truly am—a fellow human being, with no appetite for phoniness.

2. Unconditional Positive Regard. Just as it is named, this means accepting patients for who they are and eliminating any prejudices or disparaging feelings that threaten to surface. We all have personality quirks, shortcomings in communication skills, imbalances, and hidden agendas. We must not let anyone’s flaws or foibles poison our professional relationship. No matter how unpleasant, annoying, nervous, or angry our patients are, we shall respect them as unique individuals and not let them influence us in a negative, unhelpful way. Inside all of us is a yearning for respect and love. Thus, compassion is meant to be shown to all—no favoritism.

3. Empathy. Dr. Rogers believed that the therapist must be able to accurately interpret the inner emotions and struggles of the client “as if one were the person, but without ever losing the ‘as if’ condition.” Oncologists who are able to see a situation through the eyes of their patients will succeed in their mission. We must be able to “enter another’s world without prejudice,” and the best way to do this is by being perfectly comfortable in our own skin to the point that we can block our inner reactions and focus entirely on what it must be like to be the patient. Empathy will never fail to bring forth compassion.

In my opinion, compassion in the oncology clinic is 90% listening and 10% speaking, and it can only be given by those who have learned how to leave themselves out of the picture. Our opinions, biases, peculiarities, and attitudes are immaterial to the job at hand. When their lives are on the line, our patients want to know, “Does my doctor really care about me or not?” May we never be ignorant of that unspoken question, and may we always be ready to reveal the happy answer, again and again.


Thank you for this beautiful post. Nothing is more important, as Dr. Hildreth points out, than knowing “does my doctor really care about me or not?”
I have read other posts by Dr. Hildreth, and each and every time I have come away with a better understanding of what it means to be in this profession of treating cancer patients. I admire Dr. Hildreth’s philosophy so much. The first time I ever read one of his posts, I said to myself (and to some of my employees) “Dr. Hildreth is the kind of oncologist I would want to have if I ever had cancer myself”. Thank you so much, Dr. Hildreth, for being a beautiful human being and oncologist.

Irene Balowski


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Humanized Mice May Revolutionize Cancer Drug Discovery

Curator: Stephen J. Williams, Ph.D.

Humanized Mice May Revolutionize Cancer Drug Discovery

Word Cloud by Zach Day

Decades ago cancer research and the process of oncology drug discovery was revolutionized by the development of mice deficient in their immune system, allowing for the successful implantation of human-derived tumors. The ability to implant human tumors without rejection allowed researchers to study how the kinetics of human tumor growth in its three-dimensional environment, evaluate potential human oncogenes and drivers of oncogenesis, and evaluate potential chemotherapeutic therapies. Indeed, the standard preclinical test for antitumor activity has involved the subcutaneous xenograft model in immunocompromised (SCID or nude athymic) mice. More detail is given in the follow posts in which I describe some early pioneers in this work as well as the development of large animal SCID models:

Heroes in Medical Research: Developing Models for Cancer Research

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research

This strategy (putting human tumor cells into immunocompromised mice and testing therapeutic genes and /or compounds) has worked extremely well for most cytotoxic chemotherapeutics (those chemotherapeutic drugs with mechanisms of action related to cell kill, vital cell functions, and cell cycle). For example the NCI 60 panel of human tumor cell lines has proved predictive for the chemosensitivity of a wide range of compounds.

Even though the immunocompromised model has contributed greatly to the chemotherapeutic drug discovery process. using these models to develop the new line of immuno-oncology products has been met with challenges three which I highlight below with curated database of references and examples.

From a practical standpoint development of a mouse which can act as a recipient for human tumors yet have a humanized immune system allows for the preclinical evaluation of antitumoral effect of therapeutic antibodies without the need to use neutralizing antibodies to the comparable mouse epitope,   thereby reducing the complexity of the study and preventing complications related to pharmacokinetics.

Champions Oncology Files Patents for Use of PDX Platform in Immune-Oncology

Hackensack, NJ – August 17, 2015 – Champions Oncology, Inc. (OTC: CSBR), engaged in the development of advanced technology solutions and services to personalize the development and use of oncology drugs, today announced that it has filed two patent applications with the United States Patent and Trademark Office (USPTO) relating to the development and use of mice with humanized immune systems to test immune-oncology drugs and therapeutic cancer vaccines.

Dr. David Sidransky, the founder and Chairman of Champions Oncology commented, “Drug development ‎in the immune-oncology space is fundamentally changing our approach to cancer treatment. These patents represent potentially invaluable tools for developing and personalizing immune therapy based on cutting edge sequence analysis, bioinformatics and our unique in vivo models.”

Joel Ackerman, Chief Executive Officer of Champions Oncology stated, “Developing intellectual property related to our Champions TumorGraft® platform has been an important component of strategy. The filing of these patents is an important milestone in leveraging our research and development investment to expand our platform and create proprietary tools for use by our pharmaceutical partners. We continue to look for additional revenue streams to supplement our fee-for-service business and we believe these patents will help us capture more of the value we create for our customers in the future.”

The first patent filing covers the methodology used by the Company to create a mouse model, containing a humanized immune system and a human tumor xenograft, which is capable of testing the efficacy of immune-oncology agents, both as single agents and in combination with anti-neoplastic drugs. The second patent filing relates to the detection of neoantigens and their role in the development of anti-cancer vaccines.

Keren Pez, Chief Scientific Officer, explained, “In the last few years, there has been a significant increase in cancer research that focuses on exploring the power of the human immune system to attack tumors. However, it’s challenging to test immune-oncology agents in traditional animal models due to the major differences between human and murine immune systems. The Champions ImmunoGraft™ platform has the unique ability of mimicking a human adaptive immune response in the mice, which allows us to specifically evaluate a variety of cancer therapeutics that modulate human immunity.

“Therapeutic vaccines that trigger the immune system to mount a response against a growing tumor are another area of intense interest. The development of an effective vaccine remains challenging but has an outstanding curative potential. Tumors harbor mutations in DNA that result in the translation of aberrant proteins. While these proteins have the potential to provoke an immune response that destructs early-stage cancer development, often the immune response becomes insufficient. Vaccines can trigger it by proactively challenging the system with these specific mutated peptides. Nevertheless, developing anti-cancer vaccines that effectively inhibit tumor growth has been complicated, partially due to challenges in finding the critical mutations, among others difficulties. With the more recent advances in genome sequencing, it’s now possible to identify tumor-specific antigens, or neoantigens, that naturally develop as an individual’s tumor grows and mutates,” she continued.

Traumatic spinal cord injury in mice with human immune systems.

Carpenter RS, Kigerl KA, Marbourg JM, Gaudet AD, Huey D, Niewiesk S, Popovich PG.

Exp Neurol. 2015 Jul 17;271:432-444. doi: 10.1016/j.expneurol.2015.07.011. [Epub ahead of print]

Inflamm Bowel Dis. 2015 Jul;21(7):1652-73. doi: 10.1097/MIB.0000000000000446.

Use of Humanized Mice to Study the Pathogenesis of Autoimmune and Inflammatory Diseases.

Koboziev I1, Jones-Hall Y, Valentine JF, Webb CR, Furr KL, Grisham MB.

Author information


Animal models of disease have been used extensively by the research community for the past several decades to better understand the pathogenesis of different diseases and assess the efficacy and toxicity of different therapeutic agents. Retrospective analyses of numerous preclinical intervention studies using mouse models of acute and chronic inflammatory diseases reveal a generalized failure to translate promising interventions or therapeutics into clinically effective treatments in patients. Although several possible reasons have been suggested to account for this generalized failure to translate therapeutic efficacy from the laboratory bench to the patient’s bedside, it is becoming increasingly apparent that the mouse immune system is substantially different from the human. Indeed, it is well known that >80 major differences exist between mouse and human immunology; all of which contribute to significant differences in immune system development, activation, and responses to challenges in innate and adaptive immunity. This inconvenient reality has prompted investigators to attempt to humanize the mouse immune system to address important human-specific questions that are impossible to study in patients. The successful long-term engraftment of human hematolymphoid cells in mice would provide investigators with a relatively inexpensive small animal model to study clinically relevant mechanisms and facilitate the evaluation of human-specific therapies in vivo. The discovery that targeted mutation of the IL-2 receptor common gamma chain in lymphopenic mice allows for the long-term engraftment of functional human immune cells has advanced greatly our ability to humanize the mouse immune system. The objective of this review is to present a brief overview of the recent advances that have been made in the development and use of humanized mice with special emphasis on autoimmune and chronic inflammatory diseases. In addition, we discuss the use of these unique mouse models to define the human-specific immunopathological mechanisms responsible for the induction and perpetuation of chronic gut inflammation.

J Immunother Cancer. 2015 Apr 21;3:12. doi: 10.1186/s40425-015-0056-2. eCollection 2015.

Human tumor infiltrating lymphocytes cooperatively regulate prostate tumor growth in a humanized mouse model.

Roth MD1, Harui A1.

Author information



The complex interactions that occur between human tumors, tumor infiltrating lymphocytes (TIL) and the systemic immune system are likely to define critical factors in the host response to cancer. While conventional animal models have identified an array of potential anti-tumor therapies, mouse models often fail to translate into effective human treatments. Our goal is to establish a humanized tumor model as a more effective pre-clinical platform for understanding and manipulating TIL.


The immune system in NOD/SCID/IL-2Rγnull (NSG) mice was reconstituted by the co-administration of human peripheral blood lymphocytes (PBL) or subsets (CD4+ or CD8+) and autologous human dendritic cells (DC), and animals simultaneously challenged by implanting human prostate cancer cells (PC3 line). Tumor growth was evaluated over time and the phenotype of recovered splenocytes and TIL characterized by flow cytometry and immunohistochemistry (IHC). Serum levels of circulating cytokines and chemokines were also assessed.


A tumor-bearing huPBL-NSG model was established in which human leukocytes reconstituted secondary lymphoid organs and promoted the accumulation of TIL. These TIL exhibited a unique phenotype when compared to splenocytes with a predominance of CD8+ T cells that exhibited increased expression of CD69, CD56, and an effector memory phenotype. TIL from huPBL-NSG animals closely matched the features of TIL recovered from primary human prostate cancers. Human cytokines were readily detectible in the serum and exhibited a different profile in animals implanted with PBL alone, tumor alone, and those reconstituted with both. Immune reconstitution slowed but could not eliminate tumor growth and this effect required the presence of CD4+ T cell help.


Simultaneous implantation of human PBL, DC and tumor results in a huPBL-NSG model that recapitulates the development of human TIL and allows an assessment of tumor and immune system interaction that cannot be carried out in humans. Furthermore, the capacity to manipulate individual features and cell populations provides an opportunity for hypothesis testing and outcome monitoring in a humanized system that may be more relevant than conventional mouse models.

Methods Mol Biol. 2014;1213:379-88. doi: 10.1007/978-1-4939-1453-1_31.

A chimeric mouse model to study immunopathogenesis of HCV infection.

Bility MT1, Curtis A, Su L.

Author information


Several human hepatotropic pathogens including chronic hepatitis C virus (HCV) have narrow species restriction, thus hindering research and therapeutics development against these pathogens. Developing a rodent model that accurately recapitulates hepatotropic pathogens infection, human immune response, chronic hepatitis, and associated immunopathogenesis is essential for research and therapeutics development. Here, we describe the recently developed AFC8 humanized liver- and immune system-mouse model for studying chronic hepatitis C virus and associated human immune response, chronic hepatitis, and liver fibrosis.



[PubMed – indexed for MEDLINE]



Free PMC Article

Immune humanization of immunodeficient mice using diagnostic bone marrow aspirates from carcinoma patients.

Werner-Klein M, Proske J, Werno C, Schneider K, Hofmann HS, Rack B, Buchholz S, Ganzer R, Blana A, Seelbach-Göbel B, Nitsche U, Männel DN, Klein CA.

PLoS One. 2014 May 15;9(5):e97860. doi: 10.1371/journal.pone.0097860. eCollection 2014.

From 2015 AACR National Meeting in Philadelphia

LB-050: Patient-derived tumor xenografts in humanized NSG mice: a model to study immune responses in cancer therapy
Sunday, Apr 19, 2015, 3:20 PM – 3:35 PM
Minan Wang1, James G. Keck1, Mingshan Cheng1, Danying Cai1, Leonard Shultz2, Karolina Palucka2, Jacques Banchereau2, Carol Bult2, Rick Huntress2. 1The Jackson Laboratory, Sacramento, CA; 2The Jackson Laboratory, Bar Harbor, ME


  1. Paull KD, Shoemaker RH, Hodes L, Monks A, Scudiero DA, Rubinstein L, Plowman J, Boyd MR. J Natl Cancer Inst. 1989;81:1088–1092. [PubMed]
  2. Shi LM, Fan Y, Lee JK, Waltham M, Andrews DT, Scherf U, Paull KD, Weinstein JN. J Chem Inf Comput Sci. 2000;40:367–379. [PubMed]
  3. Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-Wolff A, et al. J Natl Cancer Inst. 1991;83:757–766. [PubMed]
  4. Potti A, Dressman HK, Bild A, et al. Genomic signatures to guide the use of chemotherapeutics. Nat Med. 2006;12:1294–1300. [PubMed]
  5. Baggerly KA, Coombes KR. Deriving chemosensitivity from cell lines: forensic bioinformatics and reproducible research in high-throughput biology. Ann Appl Stat. 2009;3:1309–1334.
  6. Carlson, B. Putting Oncology Patients at Risk Biotechnol Healthc. 2012 Fall; 9(3): 17–21.
  7. Salter KH, Acharya CR, Walters KS, et al. An Integrated Approach to the Prediction of Chemotherapeutic Response in Patients with Breast Cancer. Ouchi T, ed. PLoS ONE. 2008;3(4):e1908. NOTE RETRACTED PAPER

Other posts on this site on Animal Models, Disease and Cancer Include:

Heroes in Medical Research: Developing Models for Cancer Research

Guidelines for the welfare and use of animals in cancer research

Model mimicking clinical profile of patients with ovarian cancer @ Yale School of Medicine

Vaccines, Small Peptides, aptamers and Immunotherapy [9]

Immunotherapy in Cancer: A Series of Twelve Articles in the Frontier of Oncology by Larry H Bernstein, MD, FCAP

Mouse With ‘Humanized Version’ Of Human Language Gene Provides Clues To Language Development

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research

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Personalized Medicine in Cancer [Chapter 3]

Writer and Curator:  Larry H Bernstein, MD, FCAP

Personalized Medicine in Cancer

This chapter has the following ten Subsection:

3.1 The path to personalized medicine

3.2 Role of Nanobiotechnology in Developing Personalized Medicine for Cancer

3.3 The HER-2 Receptor and Breast Cancer: Ten Years of Targeted
Anti–HER-2 Therapy and Personalized Medicine

3.4 Personalized Medicine is not yet here

3.5 Biomarkers for personalized oncology: recent advances and future challenges.

3.6 Personalized oncology: recent advances and future challenges.

3.7  Pharmacogenomic biomarkers for personalized cancer treatment.

3.8 Limits to forecasting in personalized medicine: An overview

3.9 The genome editing toolbox: a spectrum of approaches for targeted modification

3.10 The Path to Personalized Medicine


3.1 The path to personalized medicine

Joanne M Meyer* and Geoffrey S Ginsburg
Current Opinion in Chemical Biology 2002, 6:434–438

Advances in personalized medicine, or the use of an individual’s molecular profile to direct the practice of medicine, have been greatly enabled through human genome research. This research is leading to the identification of a range of molecular markers for predisposition testing, disease screening and prognostic assessment, as well as markers used to predict and monitor drug response. Successful personalized medicine research programs will not only require strategies for developing and validating biomarkers, but also coordinating these efforts with drug discovery and clinical development.

The realization of personalized medicine, or the fine tailoring of the practice of medicine to an individual, is being fostered through numerous efforts aimed at characterizing individual differences in molecular processes underlying disease pathogenesis, disease progression and the response to therapeutics. Once these molecular differences are understood, therapeutic development will be enhanced by using the information to identify individuals more likely to benefit from a given intervention strategy. High-throughput genomic technologies are already providing the data that will serve as the foundation of personalized medicine.

Individual differences in the development of disease and response to therapeutics
Clearly, for many common diseases, there is abundant evidence to suggest that the molecular underpinnings of disease susceptibility, and its natural history, differ markedly among individuals. For example, while it has been demonstrated in numerous investigations that the development of obesity, asthma, type 2 diabetes and cardiovascular disease are under genetic control [1–4], there is no evidence to suggest that the genetic basis is due to variation in just a single gene. Instead, the consensus has emerged that subtle genetic differences in one or many of several genes serve as risk factors for these illnesses. Thus, while genetic variants in the melanocortin-4 receptor may explain some risk for developing obesity [5], and polymorphisms in PPARgamma may correlate with the risk of developing type 2 diabetes [6•], these variants do not explain all of these genetic diseases. There are certainly more genetic variants, or predisposition markers, to uncover. In the context of personalized medicine, the ultimate goal of these types of studies is to provide a suite of markers that can be used to assess one’s lifetime risk of developing disease in the presence of various environmental (e.g. diet, lifestyle) variables.

As with disease predisposition, individual differences characterize disease progression. For example, some individuals with impaired glucose tolerance will proceed quite rapidly to type 2 diabetes, whereas others proceed slowly. Similarly, individuals diagnosed with rheumatoid arthritis may or may not develop erosive disease. In both of these cases, genetic variation, that is, variation measured at the DNA level, may be a good predictor of the individual differences that emerge as disease progresses. For example, Brinkman et al. [7] have demonstrated that a polymorphism in TNF-α correlates with erosive rheumatoid arthritis, but shows no association with non-erosive disease. Alternatively, variation in disease progression may be best predicted by a combination of genetic and environmental factors, the impact of which is indexed through changes in gene expression in relevant tissues, or changes in secreted protein levels in serum or synovial fluid. In our laboratories, we are using a range of genomics technologies to find markers for disease progression that are both stable (DNA) as well as dynamic (mRNA, protein), giving us the opportunity to evaluate the utility of both types of markers in prospective studies.

Given that individual variability in disease predisposition and progression exists and has the potential of being molecularly characterized, it is not at all surprising that such differences also characterize response to therapeutics (see Figure 1). Marked individual variation in the efficacy and toxicity of therapeutic compounds is common and can have a profound impact on the success of a pharmaceutical clinical development program. Clearly, molecular markers that predict the variation in these endpoints could be extremely useful in clinical trials, drug development and clinical practice, as they would allow the identification of patients who would benefit most from the drug.

Technological advances drive broad biomarker discovery. While the existence of individual differences in disease predisposition, progression and response to therapeutics is far from a novel concept, our ability to comprehensively measure the molecular markers that track these processes, and draw proper inferences from large amounts of molecular data, is novel. Over the past decade, significant advancements have been made in technologies to discover variation at the mRNA, DNA and protein levels. Indeed, with the advent of glass and nylon microarray technologies for gene-expression studies, it is quite feasible to characterize the expression levels of 30 000 genes in tissue samples from dozens, if not hundreds, of individuals. Certainly, several years ago, although it would have been theoretically possible to assess this number of genes using northern blot analysis, it never would have been undertaken in a sample from even a single individual. In the same fashion, highthroughput technologies for DNA polymorphism discovery and single nucleotide polymorphism (SNP) genotyping, coupled with broad academic and commercial initiatives to characterize genetic variation genome-wide [8•], are resulting in catalogs of variants that can be used in large-scale experiments. To complement these efforts, searches for ‘haplotype blocks’, or correlated patterns of SNPs that can be adequately represented by fewer SNPs, are underway and have the promise of reducing the amount of genotyping required for genome-wide searches [9•,10•]. For proteinbased discovery initiatives, traditional 2D electrophoresis experiments are used in conjunction with advanced mass spectrometry to discover protein markers in a range of complex fluids, including serum, plasma, synovial fluid and cerebral spinal fluid.

Coupled with the advent of these technologies have been extensive efforts to collect appropriate tissues and fluids for mRNA, DNA and protein analysis. These collections have been part of pharmaceutical clinical trials, as well as clinical studies established for the purpose of characterizing biomarkers. The latter studies may involve small numbers of patient samples for initial biomarker discovery efforts, as well as large-scale, disease registry initiatives designed to evaluate and, in some cases, prospectively validate, biomarkers in the relevant patient populations.

Figure 1  (not shown) The role of molecular biomarkers in disease management. Areas where molecular biomarkers will benefit personalized medicine include disease predisposition, screening and prognosis, as well as drug response and drug monitoring. The nature of the markers (DNA, mRNA or protein) will vary with the disease and the stage of their application. Rx indicates treatment.

The predictive value of biomarkers The impact that advanced genomic technologies and carefully designed biomarker studies will have on the personalization of medicine is foreshadowed in the current literature. For example, Mallal et al. [13•] conducted a pharmacogenetic investigation (i.e. a genetic study of drug response) of abacavir, an HIV-1 nucleoside reverse transcriptase inhibitor. They implicated MHC alleles that predict response to hypersensitivity among 5% of the HIV cases receiving the drug. Their findings suggest that screening patients for the presence of the predisposing MHC haplotype could reduce the prevalence of hypersensitivity to abacavir from 9% to 2.5%. While this study is small in scale in its characterization of genetic variation, it adds to the existing literature on several other variants (including those in MDR1, the multidrug transporter P-glycoprotein and CYP2D6, a cytochrome P450 isoszyme) that correlate with the pharmacokinetic (drug clearance) characteristics of protease inhibitors and non-nucleoside reverse transcriptase inhibitors [14]. Additionally, genetic polymorphisms in chemokines and chemokine receptors (including RANTES, MIP-1α and CCR5) have been found to correlate with both the susceptibility to HIV-1 infection and the progression of disease [15•]. Taken together, these findings may lead to the development of a panel of polymorphisms that would personalize HIV therapy, by determining when to initiate therapy and how to choose compounds that will maximize efficacy and minimize adverse effects.

Figure 2 (not shown) Personalized medicine — integrating drug discovery and development through molecular medicine. Genomically derived biomarkers are being identified throughout the drug discovery and clinical development process. They will not only support personalized medicine, but will also enhance drug discovery and clinical development by generating new targets, validating targets and identifying patients that will benefit from novel therapeutics.

Pharmacogenetic efforts have also successfully characterized polymorphisms that correlate with response to asthma therapeutics. For example, Drazen et al. [16•] showed that a promotor polymorphism in 5-lipoxygenase, which alters transcription levels of the gene, also correlates with response to a derivative of the drug Zileuton, a 5-lipoxygenase inhibitor. Of the individuals who did not respond to Zileuton, 20% carried rare variant alleles at this locus. By contrast, all of the responders had wild-type alleles. Similarly in a study of genetic polymorphisms of the β-adrenergic receptor, Drysdale et al. [17•] demonstrated that a haplotype, or SNP signature across the gene, correlated strongly with asthma patients’ response to β-agonists. These two examples again demonstrate the possibility of using an individual’s genotype to suggest a therapeutic strategy that is more likely to be efficacious. Certainly, before such tests are incorporated into clinical practice, additional genetic markers would have to be coupled with the existing polymorphisms to make the resulting tests highly sensitive and specific.

In addition to these DNA-based strategies, recent applications of proteomics and expression profiling have generated a range of screening, prognostic and drug-response or ‘pharmacogenomic’ biomarkers. Many advances in the use of these technologies have been in oncology, where there is a tremendous need for serum-based screening markers and where tissue samples for expression profiling studies are easily obtained. For example, Petricoin et al. [18•] demonstrated that proteomic spectra, derived from a mass spectrometry analysis of serum, could be used to distinguish women with ovarian cancer from unaffected women. Indeed, the protein markers on a ‘training set’ of 100 samples and a validation set of 110 additional samples, had a sensitivity of 100% and a specificity of 94%. These encouraging results suggest that a serum-based protein assay may indeed become a viable mode of ovarian cancer screening in the general population. mRNA strategies for identifying prognostic markers for cancers have also proved successful. For example, in our collaborative studies [19•], we have shown that Melastatin, a melanocyte-specific gene identified through a genomics analysis of benign and malignant melanoma, is an effective prognostic marker for cutaneous malignant melanoma. In this work, uniform melastatin mRNA expression correlated strongly with disease-free survival, even after adjusting for other prognostic factors. In a similar fashion, mRNA strategies have generated pharmacogenomic markers for ovarian cancer. Hartmann et al. [20] studied the expression of 30 000 human genes in 51 tumors that were sensitive and resistant to platinum–paclitaxel chemotherapy and identified a subset of 10 markers that were highly predictive of outcome in an independent sample of tumors. Overall, these examples of biomarker studies in oncology demonstrate the broad application such markers will have for cancer screening, prognosis and response to therapeutics.

Turning biomarker discoveries into personalized medicines All of the examples cited provide excellent demonstrations of the power of new technologies to deliver a range of biomarkers that index individual differences in disease predisposition, progression and response to therapeutics. Thus, they clearly form a basis for the ‘personalization’ of medicine. However, the discovery of these markers is not sufficient for the pharmaceutical industry to deliver personalized medicines. Indeed, the delivery of such medicines will require the careful integration of biomarker discovery and validation programs into drug discovery and clinical development programs (see Figure 2). This integration will serve two key purposes. First, and foremost, by initiating SNP, expression profiling and proteomics biomarker programs early on in the drug discovery process, one can carefully weave the discovery and validation of biomarkers into drug discovery and development timelines; the risk of ‘retro-fitting’ biomarker programs to a clinical trial would be avoided.

Conclusions Clearly, several challenges remain to achieve a successful integration of large-scale, biomarker studies with drug development. While there has been an incredible advance in high-throughput, molecular technologies, over the past several years, further improvements in technologies and validation strategies are required to capture the true extent of individual differences in molecular markers. For example, although it is plausible to consider screening the genome for SNPs or haplotypes that correlate with disease pre-disposition or drug response, the current cost of SNP genotyping makes this impractical. Additionally, bioinformatic and statistical advances are needed to extract the most relevant data from the wealth of molecular information generated by new technologies, and these advances must be effectively communicated to the heath-care environment. Finally, and most importantly, plans must be in place to provide adequate validation for the enormous number of candidate biomarkers that will emerge from the studies. Validation will require access to large, and in some cases, prospective, collections of well annotated clinical samples with appropriate consent and security issues addressed. While these issues, as well as the commercial and regulatory considerations around the development of personalized medicines, are indeed challenging, the successful execution of biomarker programs will have an enormous impact on our ability to tailor medical practice to the individual.

3.2 Role of Nanobiotechnology in Developing Personalized Medicine for Cancer

K. K. Jain
Technol Cancer Res Treat Dec 2005; 4(6): 645-650


Personalized medicine simply means the prescription of specific therapeutics best suited for an individual. Personalization of cancer therapies is based on a better understanding of the disease at the molecular level. Nanotechnology will play an important role in this area. Nanobiotechnology is being used to refine discovery of biomarkers, molecular diagnostics, drug discovery and drug delivery, which are important basic components of personalized medicine and are applicable to management of cancer as well. Examples are given of the application of quantum dots, gold nanoparticles, and molecular imaging in diagnostics and combination with therapeutics – another important feature of personalized medicine. Personalized medicine is beginning to be recognized and is expected to become a part of medical practice within the next decade. Personalized management of cancer, facilitated by nanobiotechnology, is expected to enable early detection of cancer, more effective and less toxic treatment increasing the chances of cure.

3.3 The HER-2 Receptor and Breast Cancer: Ten Years of Targeted Anti–HER-2 Therapy and Personalized Medicine

Jeffrey S. Ross,  Elzbieta A. Slodkowska,  W. Fraser Symmans, et al.
The Oncologist 2009; 14:320 –368


  1. Contrast the current strengths and limitations of the three main slide-based techniques (IHC, FISH, and CISH) currently in clinical use for testing breast cancer tissues for HER-2 status.
  2. Compare the efficacy of trastuzumab- and lapatinib-based regimens in the adjuvant and metastatic settings as reported in published clinical trials and regulatory approval databases.
  3. Contrast the list of biomarkers that have been associated with clinical resistance to trastuzumab and lapatinib and describe their current level of validation.

The human epidermal growth factor receptor (HER-2) oncogene encodes a transmembrane tyrosine kinase receptor that has evolved as a major classifier of invasive breast cancer and target of therapy for the disease. The validation of the general prognostic significance of HER-2 gene amplification and protein overexpression in the absence of anti–HER-2 targeted therapy is discussed in a study of 107 published studies involving 39,730 patients, which produced an overall HER-2– positive rate of 22.2% and a mean relative risk for overall survival (OS) of 2.74. The issue of HER-2 status in primary versus metastatic breast cancer is considered along with a section on the features of metastatic HER- 2–positive disease. The major marketed slide-based HER-2 testing approaches, immunohistochemistry, fluorescence in situ hybridization, and chromogenic in situ hybridization, are presented and contrasted in detail against the background of the published American Society of Clinical Oncology–College of American Pathologists guidelines for HER-2 testing. Testing issues, such as the impact of chromosome 17 polysomy and local versus central HER-2 testing, are also discussed. Emerging novel HER-2 testing techniques, including mRNA-based testing by real-time polymerase chain reaction and DNA microarray methods, HER-2 receptor dimerization, phosphorylated HER-2 receptors, and HER-2 status in circulating tumor cells, are also considered. A series of biomarkers potentially associated with resistance to trastuzumab is discussed with emphasis on the phosphatase and tensin homologue deleted on chromosome ten/Akt and insulin-like growth factor receptor pathways. The efficacy results for the more recently approved small molecule HER- 1/HER-2 kinase inhibitor lapatinib are also presented along with a more limited review of markers of resistance for this agent. Additional topics in this section include combinations of both anti–HER-2 targeted therapies together as well as with novel agents including bevacizumab, everolimus, and tenespimycin. A series of novel HER-2–targeting agents is also presented, including pertuzumab, ertumaxomab, HER-2 vaccines, and recently discovered tyrosine kinase inhibitors. Biomarkers predictive of HER-2 targeted therapy toxicity are included, and the review concludes with a consideration of HER-2 status in the prediction of response to non–HER-2 targeted treatments including hormonal therapy, anthracyclines, and taxanes.

Biology, Pathology, Diagnosis, And Clinical Significance Of Her-2–Positive Breast Cancer

The human epidermal growth factor receptor 2 (HER-2, HER-2/neu, c-erbB-2) gene, first discovered in 1984 by Weinberg and associates [1], is localized to chromosome 17q and encodes a transmembrane tyrosine kinase receptor protein that is a member of the epidermal growth factor receptor (EGFR) or HER family (Fig. 1) [2]. This family of receptors is involved in cell– cell and cell–stroma communication primarily through a process known as signal transduction, in which external growth factors, or ligands, affect the transcription of various genes, by phosphorylating or dephosphorylating a series of transmembrane proteins and intracellular signaling intermediates, many of which possess enzymatic activity. Signal propagation occurs as the enzymatic activity of one protein turns on the enzymatic activity of the next protein in the pathway [3]. Major pathways involved in signal transduction, including the Ras/mitogen-activated protein kinase pathway, the phosphatidylinositol 3 kinase (PI3K)/Akt pathway, the Janus kinase/signal transducer and activator of transcription pathway, and the phospholipase C pathway, ultimately affect cell proliferation, survival, motility, and adhesion. Receptor activation requires three variables, a ligand, a receptor, and a dimerization partner [4]. After a ligand binds to a receptor, that receptor must interact with another receptor of identical or related structure in a process known as dimerization in order to trigger phosphorylation and activate signaling cascades. Therefore, after ligand binding to an EGFR family member, the receptor can dimerize with various members of the family (EGFR, HER-2, HER-3, or HER-4). It may dimerize with a like member of the family (homodimerization) or it may dimerize with a different member of the family (heterodimerization). The specific tyrosine residues on the intracellular portion of the HER-2/neu receptor that are phosphorylated, and hence the signaling pathways that are activated, depend on the ligand and dimerization partner. The wide variety of ligands and intracellular crosstalk with other pathways allow for significant diversity in signaling. Although no known ligand for the HER-2 receptor has been identified, it is the preferred dimerization partner of the other family members. HER-2 heterodimers are more stable [5, 6] and their signaling is more potent [7] than receptor combinations without HER-2. HER-2 gene amplification and/or protein overexpression has been identified in 10%–34% of invasive breast cancers [1]. Unlike a variety of other epithelial malignancies, in breast cancer, HER-2 gene amplification is uniformly associated with HER-2 (p185neu) protein overexpression and the incidence of single copy overexpression is exceedingly rare [8]. HER-2 gene amplification in breast cancer has been associated with increased cell proliferation, cell motility, tumor invasiveness, progressive regional and distant metastases, accelerated angiogenesis, and reduced apoptosis [9].When classified by routine clinicopathologic parameters and compared with HER-2– negative tumors, HER-2–positive breast cancer is more often of intermediate or high histologic grade, more often lacking estrogen receptors (ERs) and progesterone receptors (PgRs) (ER and PgR negative), and featuring positive lymph node metastases at presentation [1]. In the recent molecular classification of breast cancer, positive HER-2 status does not constitute a unique molecular category and is identified in both the “HER-2” and “luminal” tumor classes [10].

Figure 1 (not shown)

Figure 1. The human epidermal growth factor receptor (HER) gene family. This image depicts the complex crosstalk between members of the HER family of receptor tyrosine kinases and intracellular signaling. Activated HER receptors can function to both stimulate and inhibit downstream signaling of members of other biologic pathways. Note that HER-2 has no activating ligands and HER-3 lacks a tyrosine kinase domain. HER-2–mediated signaling is associated with cell proliferation, motility, resistance to apoptosis, invasiveness, and angiogenesis. The figure shows the complexity of signaling pathways initiated by, and influenced by, HER family protein receptors at the cell surface.

HER-2 Status and Prognosis in Breast Cancer Both morphology-based and molecular-based techniques have been used to measure HER-2/neu status in breast cancer clinical samples [11–117]. By a substantial majority, abnormalities in HER-2 expression at the gene, message, or protein level have been associated with adverse prognosis in both lymph node–negative and lymph node–positive breast cancer. Of the 107 studies considering 39,730 patients listed in Table 1, 95 (88%) of the studies determined that either HER-2 gene amplification or HER-2 (p185 neu) protein overexpression predicted breast cancer outcome on either univariate or multivariate analysis. In 68 (73%) of the 93 studies that featured multivariate analysis of outcome data, the adverse prognostic significance of HER-2 gene, message, or protein overexpression was independent of all other prognostic variables. In only 13 (12%) of the studies, no correlation between HER-2 status and clinical outcome was identified. Of these 13 noncorrelating studies, eight (62%) used immunohistochemistry (IHC) on paraffin-embedded tissues as the HER-2/protein detection technique, two (15%) used fluorescence in situ hybridization (FISH), two (15%) used Southern analysis, and one (7%) used a real-time polymerase chain reaction (RT-PCR) technique. Of the 15 studies that used the FISH technique, 13 (87%) showed univariate prognostic significance of gene amplification, and 11 of these (85%) showed prognostic significance on multivariate analysis as well. The two studies that used chromogenic in situ hybridization (CISH) HER-2 gene amplification detection techniques both found that HER-2 amplification was an independent predictor of outcome on multivariate analysis [100, 112]. However, interpretation of these studies is complicated by the fact that most studies included patients who received variable types of systemic adjuvant therapy; therefore, the pure prognostic value of HER-2 overexpression in the absence of any systemic adjuvant therapy is incompletely understood.

Table 1 HER-2 status and prognosis in breast cancer (not shown)

HER-2 Positivity Rates The frequency of HER-2 positivity in all of the studies presented in Table 1 was 22.2%, with a range of 9%–74%. The HER-2–positive rate was similar for IHC, at 22% (range, 10%–74%), and FISH, at 23.9% (range, 14.7%– 68%). In current practice, HER-2–positive rates have trended below 20%, with most investigators currently reporting that the true positive rate is in the range of 15%–20%. The HER-2– positive rate may be higher when metastatic lesions are tested, and tertiary hospitals and cancer centers report slightly higher rates than community hospitals and national reference laboratories. Relative Risk and Hazard Ratio In Table 1, a number of studies provided data as to the relative risk (RR) of untreated HER-2–positive breast cancer being associated with an adverse clinical outcome. For OS, the mean RR was 2.74 (range, 1.39 – 6.93) and the median was 2.33; for disease-free survival (DFS), the mean RR was 2.04 (range, 1.30 –3.01) and the median was 1.8. In several studies, the RR was estimated with a hazard ratio (HR) model. The mean HR was 2.12 (range, 1.6 –2.7) and the median was 2.08. HER-2 Expression and Breast Pathology The association of HER-2–positive status with specific pathologic conditions of the breast is summarized in Table 2. HER-2 overexpression has been consistently associated with higher grades and extensive forms of ductal carcinoma in situ (DCIS) and DCIS featuring comedo-type necrosis [118 –121]. The incidence of HER-2 positivity in DCIS has varied in the range of 24%–38% in the published literature, which appears to be slightly higher than that for invasive breast cancer [118 –121]. Routine testing for HER-2 status in DCIS is not widely performed. However, should anti– HER-2 targeted therapies directed at HER-2–positive DCIS result in a reduction in the development of invasive disease, the widespread use of HER-2 testing in DCIS would be adopted. Finally, the invasive carcinoma that develops in association with HER-2–positive DCIS may, on occasion, not feature a HER-2–positive status, a finding that has led investigators to believe that HER-2 gene amplification may not be required for the local progression of breast cancer [122]. Compared with invasive ductal carcinoma (IDC), HER-2 gene amplification occurs at a significantly lower rate in invasive lobular carcinoma (ILC) (10%), but has also been linked to an adverse outcome [85]. HER-2 positivity is linked exclusively to the pleomorphic variant of ILC and is not encountered in classic ILC [123]. HER-2 amplification is strongly correlated with tumor grade in both IDC and ILC. For example, in one study, only one of 73 grade I IDC cases and one of 67 low-grade classic ILC cases showed HER-2 amplification detected by FISH [86]. HER-2 overexpression and HER-2 amplification have been a consistent feature of both mammary and extramammary Paget’s disease [124, 125] (Fig. 2). HER-2 amplification and HER-2 overexpression have been associated with adverse outcome in some studies of male breast carcinoma [126 –129], but not in others [130 –132]. The incidence of HER-2 positivity appears to be lower in male breast cancer than in female breast cancer [126 –132]. Documented responses in male breast cancer to HER-2–targeting agents have been described, and therefore treatment with trastuzumab is an acceptable option for these patients, but the true activity rate remains uncertain [133]. The rate of HER-2 overexpression in mucinous (colloid) breast cancers is extremely low, although, on occasion, it has been associated with aggressive disease [134 –136]. In medullary breast carcinoma, HER-2 testing has consistently found negative results [137]. Similarly, HER-2 positivity is extremely rare in cases of tubular carcinoma [138]. HER-2 status has not been consistently linked to the presence of inflammatory breast cancer [139, 140]. Molecular studies of hereditary breast cancer including cases with either BRCA1 or BRCA2 germline mutations have found a consistently lower incidence of HER-2–positive status for these tumors [141].

Figure 2 not shown

Figure 2. Human epidermal growth factor receptor (HER)-2–positive Paget’s disease of the nipple. In this patient, who presented with HER-2–positive invasive duct carcinoma, classic clinical features of Paget’s disease of the nipple were present. A section of the nipple from the mastectomy specimen shows 3+ continuous cell membrane immunoreactivity for HER-2 protein. Nearly 100% of Paget’s disease of the breast cases are HER-2 positive (see text).

Breast sarcomas and phyllodes tumors have consistently been HER-2 negative [142]. Finally, low-level HER-2/neu overexpression has been identified in benign breast disease biopsies and is associated with a greater risk for subsequent invasive breast cancer [143].

HER-2 Status in Primary Versus Metastatic Breast Cancer The majority of studies that have compared the HER-2 status in paired primary and metastatic tumor tissues have found an overwhelming consistency in the patient’s status regardless of the method of testing (IHC versus FISH) [144 –151]. However, several recent studies indicated 20%–30% discordance rates between the HER-2 status of primary and metastatic lesions. Some of these studies have featured relatively high HER-2–positive rates on both paired specimens (> 35% positive), which has created concern about the conclusions of these reports [152]. Also, considering that 10%–30% discordance rates have been reported even when the same tumor is tested repeatedly, it remains uncertain if the discordance rates seen between primary and metastatic sites is higher than expected by the less than perfect reproducibility of the various HER-2 assays. Increasingly, emerging data suggest that there are changes in HER-2 expression between primary and metastatic disease. This is particularly true after intervening HER-2– directed therapy, but also happens in the absence of such treatment. In cases where the original primary HER-2 test result is questioned because of technical or interpretive issues and in patients where there has been an unusually long (i.e., > 5-year) interval between the primary occurrence and the detection of metastatic disease, retesting of a metastatic lesion may be warranted. Thus, although routine HER-2 testing of metastatic disease is advocated by some investigators, the preponderance of data indicates that the HER-2 status remains stable and that routine retesting of HER-2 may not be needed for most patients with metastatic disease.

Features of Metastatic HER-2–Positive Breast Cancer Metastatic HER-2–positive breast cancer retains the phenotype of the primary tumor not only in HER-2 status, but also is typically ER/PgR negative, moderate to high tumor grade, DNA aneuploid with high S phase fraction, and featuring ductal rather than lobular histology. In the era prior to the initiation of HER-2–targeted therapy, HER-2–positive breast cancer was more likely to spread early to major visceral sites including the axillary lymph nodes, bone marrow, lungs, liver, adrenal glands, and ovaries [153]. In the post–HER-2 targeted therapy era, the incidence of progressive visceral metastatic disease in HER-2–positive tumors has diminished and has frequently been superseded by the development of clinically significant central nervous system (CNS) metastatic disease [154 –157]. It is widely held that the success in the control of visceral disease with trastuzumab has unmasked previously occult CNS disease and, because of the inability of the therapeutic antibody to cross the blood– brain barrier, allowed brain metastases to progress during the extended OS duration of treated patients [154, 155]. The small-molecule drug lapatinib has shown some promise for targeting HER-2–positive CNS metastases that are resistant to trastuzumab-based therapies in initial studies [158].

Interaction of HER-2 Expression with Other Prognosis Variables HER-2 gene amplification and protein overexpression have been associated consistently with high tumor grade, DNA aneuploidy, high cell proliferation rate, negative assays for nuclear protein receptors for estrogen and progesterone, p53 mutation, topoisomerase IIa amplification, and alterations in a variety of other molecular biomarkers of breast cancer invasiveness and metastasis [159 –161].

Figure 3. Human epidermal growth factor receptor (HER)-2 testing.
(not shown)  (A): Immunohistochemistry (IHC). This panel depicts the four categories of HER-2 IHC staining including 0 and 1+ (negative), 2+ (equivocal), and 3+ (positive) using the American Society of Clinical Oncology–College of American Pathologists guidelines for HER-2 IHC scoring. (B): Fluorescence in situ hybridization (FISH). This panel demonstrates a case of invasive duct carcinoma, on the left, negative for HER-2 gene amplification (gene copy number < 4) and a case of HER-2 gene–amplified breast cancer (gene copy number > 6),

FISH. The FISH technique (Fig. 3B), like IHC, is a morphology-driven slide-based DNA hybridization assay using fluorescent-labeled probes. Both the hybridization steps and the slide scoring can be automated. FISH has the advantages of a more objective scoring system and the presence of a built-in internal control consisting of the two HER-2 gene signals present both in benign cells and in malignant cells that do not feature HER-2 gene amplification.

IHC Versus FISH. Although the FISH method is more expensive and time-consuming than IHC, numerous studies have concluded that this cost is well borne by the greater accuracy and more precise use of anti–HER-2 targeted therapies [179 –180, 182–183]. FISH is considered to be more objective and reproducible in a number of systematic reviews [165, 180, 183–186]. In one study, the concordance rates between IHC and FISH were highest in tumors scored by IHC as 0 and 1+ and lowest for 2+ and 3+ cases [183]. Currently, the majority (approximately 80%) of HER-2 testing in the U.S. commences with a screen by IHC, with results of 0 and 1+ considered negative, 2+ considered equivocal and referred for FISH testing, and 3+ considered positive. In a pharmacoeconomic study of patients being considered for trastuzumab-based treatment for HER-2– positive tumors, FISH was found to be a cost-effective diagnostic approach “from a societal perspective” [187].

CISH and Silver In Situ Hybridization. The CISH method (Fig. 3E) and silver in situ hybridization (SISH) method feature the advantages of both IHC (routine microscope, lower cost, familiarity) and FISH (built-in internal control, subjective scoring, the more robust DNA target) [190, 191]. The CISH technique uses a single HER-2 probe, detects HER-2 gene copy number only, and was recently approved by the FDA to define patient eligibility for trastuzumab treatment. The SISH method employs both HER-2 and chromosome 17 centromere probes hybridized on separate slides and is currently under review by the FDA. Numerous studies have confirmed a very high concordance between CISH and FISH, typically in the 97%–99% range [191–203]. Similar to FISH, CISH has its highest correlation with IHC 0, 1+, and 3+ results and lowest correlation with IHC 2+ staining.

Chromosome 17 Polysomy. The incidence of chromosome 17 polysomy has varied from as low as 4% to as high as 30% in studies of invasive breast cancer [204 –208]. This may reflect differences in the definition of polysomy ranging from a low-level definition of more than two copies per cell to a high of more than four copies per cell of the chromosome. Most studies have linked chromosome 17 polysomy with greater HER-2 protein overexpression [204 –207], but some have found that protein overexpression only occurs in the presence of selective HER-2 gene amplification [204].

The 2007 ASCO-CAP Guidelines. In early 2007, a combined task force from ASCO and the CAP issued a series of recommendations designed to improve the accuracy of tissue-based HER-2 testing in breast cancer [212]. A summary of the ASCO-CAP guidelines is provided in Table 4. Highlights of these recommendations include (a) standardizing fixation in neutral-buffered formalin for no less than 6 hours and no more than 48 hours, (b) unlike their respective FDA-approval specifications, defining equivocal zones for the IHC, FISH, and CISH tests, (c) establishing a standardized quality assurance program for testing laboratories, and (d) requiring the participation of these laboratories in a proficiency testing program [212]. The published guidelines were designed to improve the overall precision and reliability of all types of slide-based HER-2 tests and remained neutral as to the relative superiority of one test over the others.

Figure 4. Real-time polymerase chain reaction (RT-PCR). In this RT-PCR assay using the Taqman RT-PCR System (Applied Biosystems Inc., Foster City, CA), note the detection of increased human epidermal growth factor receptor(HER)-2 mRNA expression in green detected at lower numbers of amplification cycles compared with the two housekeeping genes shown in red and blue.

Figure 5. DNA microarray. In this image, increased expression of human epidermal growth factor receptor (HER)-2 mRNA has been detected using a proprietary DNA microarray system (Millennium Pharmaceuticals, Inc., Cambridge, MA). The microarray demonstrates the coexpression of seven genes (HER-2 is second from the bottom) related to the amplification of HER-2 DNA in this case of HER-2–positive breast cancer.

Her-2–Targeted Therapy and the Treatment of Her-2–Positive Breast Cancer

Trastuzumab: HER-2 Testing and the Prediction of Response to Trastuzumab Therapy Using recombinant technologies, trastuzumab (Herceptin; Genentech, South San Francisco, CA), a monoclonal IgG1 class humanized murine antibody, was developed by the Genentech Corporation to specifically bind the extracellular portion of the HER-2 transmembrane receptor. This antibody therapy was initially targeted specifically for patients with advanced relapsed breast cancer that overexpresses HER-2 protein [262]. Since its launch in 1998, trastuzumab has become an important therapeutic option for patients with HER-2–positive breast cancer and is widely used for its approved indications in both the adjuvant and metastatic settings (Fig. 6) [185, 263–265]. Although trastuzumab is approved as a single-agent regimen, most patients are treated with trastuzumab plus cytotoxic agents. Table 5 summarizes the significant clinical trials that contributed to the regulatory approvals of trastuzumab.

This topic is scheduled for another article.

Trastuzumab Combinations. Since the FDA approval in 1998 of two trastuzumab plus chemotherapy combinations, a number of additional approaches have gained favor in the clinical practice community. The National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines [284] currently recommend the following regimens for the first-line treatment of HER-2–positive MBC: trastuzumab plus single agents— either paclitaxel (every 3 weeks or weekly), docetaxel (every 3 weeks or weekly), or vinorelbine (weekly). For combination therapies, the NCCN recommends trastuzumab plus paclitaxel and carboplatin (every 3 weeks) or docetaxel plus carboplatin. Recently, carboplatin-based trastuzumab combinations have gained interest as a result of both the apparent boost in efficacy as measured by a higher overall response rate and longer progression-free survival time and the cardioprotective benefits of avoiding an anthracycline-containing regimen [285].

Neoadjuvant Setting The results of trastuzumab-based neoadjuvant studies (Table 5) have received significant recent interest in the oncology community [289]. Virtually all completed and in progress clinical trials have demonstrated a significant enhancement in the rate of pathologic complete response (pCR), the primary endpoint in these studies, in cases of patients with HER-2–positive breast cancer that received trastuzumab in the neoadjuvant setting [290 –297]. This benefit of the addition of trastuzumab in the neoadjuvant setting appears to be independent of, if not enhanced by, the coexistence of ER positivity [297]. Among the potential explanations for the apparent greater chemosensitivity of HER-2–positive tumors cotreated with trastuzumab in the neoadjuvant setting is the concept that HER-2 gene amplification is in some way related to the growth and survival of breast cancer stem cells [298, 299].

Biomarkers of Trastuzumab Resistance Since trastuzumab was introduced for the treatment of MBC in 1998, there has been growing interest in the discovery and potential clinical utility of biomarkers designed to predict resistance to the drug. Current approaches to HER-2 testing provide a negative predictor of drug response: the test does not predict which patients will respond to trastuzumab, it predicts which patients are unlikely to benefit.

Neoadjuvant Setting The Neo-ALTTO trial is a randomized, open-label, multicenter, phase III study comparing the efficacy of neoadjuvant lapatinib plus paclitaxel with that of trastuzumab plus paclitaxel and with concomitant lapatinib and trastuzumab plus paclitaxel given as neoadjuvant treatment in HER-2– positive primary breast cancer [337].

Biomarkers of Lapatinib Resistance In that lapatinib was approved 9 years after trastuzumab, considerably less information has been published concerning markers of efficacy or resistance to the drug [331, 341– 343].

Trastuzumab Since its introduction in the MBC setting and continuing throughout its advance into use in both the adjuvant and neoadjuvant settings, trastuzumab has been associated with the development of a variety of toxicities [384]. In the original registration trial for MBC, trastuzumab was associated with a variety of adverse events, including pain, gastrointestinal disturbances, minor hematologic deficiencies, pulmonary symptoms, and congestive heart failure (CHF) [265]. Cardiac toxicity has remained the most significant limiting factor for the use of trastuzumab [384 –389]. A major consideration in the development of cardiac toxicity in patients treated with trastuzumab has been their prior or concomitant exposure to anthracycline drugs, also associated with dose-dependent irreversible heart damage [384 – 389].

Lapatinib The most frequent adverse reactions in the lapatinib– capecitabine registration trial for MBC combination were diarrhea (65%), palmar–plantar erythrodysesthesia (53%), nausea (44%), rash (28%), vomiting (26%), and fatigue (23%) [332]. In a comprehensive analysis of the clinical trials featuring lapatinib in combination with various other agents, the overall incidence of LVEF decline was 1.6%, with 0.2% of patients experiencing symptomatic CHF [389].

HER-2 Status and the Prediction of Response to Non–HER-2 Targeted Therapy The use of HER-2 status to predict responsiveness or resistance to hormonal therapies, advocated by a number of oncologists, remains controversial. It has been reported that ER-positive/HER-2–positive patients are either less responsive or completely resistant to single-agent tamoxifen [391–393]. When measured as continuous variables, the expression of HER-2 appears to be inversely related to the expression of ER and PgR even in hormone receptor–positive tumors [394].

Anthracyclines HER-2 overexpression has also been associated with enhanced response rates to anthracycline-containing chemotherapy regimens in most, but not all, studies [42, 410 – 414].

Radiation Therapy Initially, in the era prior to the introduction of anti–HER-2 targeted therapy, HER-2–positive status was associated with a higher rate of local recurrence in some studies of breast cancer treated with surgery and radiation therapy alone, but not in others [427– 429]. However, although large-scale, randomized, prospective studies are lacking, HER-2–positive tumors treated with trastuzumab-based neoadjuvant chemotherapy combined with external-beam radiation have indicated a favorable response in locally advanced breast cancer [430].

Summary The history of the discovery of the HER-2 oncogene in an animal model in 1984, the translation of this finding to the clinical behavior of human breast cancer, and the introduction of the first anti-HER targeted therapy in 1998 is clearly a triumph of “bench to bedside” medicine. In the 10 years that have now passed since the regulatory approval of the first anti–HER-2 targeted therapy, trastuzumab, thousands of preclinical and clinical studies have considered HER-2 as a prognostic factor, its ability to predict response to hormonal and cytotoxic treatments, the best way to test for it in routine specimens, and the clinical efficacy of targeting it in a wide variety of clinical settings. Given the proven efficacy of trastuzumab and lapatinib for the treatment of MBC, and also in the adjuvant and neoadjuvant settings, the critical issue as to which test (IHC versus FISH versus CISH versus mRNA based) is the most accurate and reliable method to determine HER-2 status in breast cancer has continued to increase in importance.

3.4 Personalized Medicine is not yet here

ESMO Personalized Medicine
Written by Dr Marina Garassino for ESMO

The aim of personalised medicine is clearly to make therapy more efficient for patients. A very, very small step in the process is to try to identify for every patient the main molecular driver of their tumour. We have to understand that patients differ between each other, although they may have the same cancer type; for example, every patient with breast cancer or bowel cancer will have a unique tumor. This is entirely new knowledge, so what we are trying to do now in the medical community is to identify for each patient his/ her type of disease and then to give the drug that will work best. We are moving forward with an incredible amount of new data and innovative knowledge on genetic characteristics and subsequent proteomic changes* in the tumor. The challenge is now about how to exploit this information in order to offer targeted treatment and generally improve patient care.

For a number of years we have classified tumors according to their site of origin and using a classification system called “TNM”. Researchers and clinicians once thought that all cancers that derived from the same site were biologically similar and they differed perhaps only in their pathohistological* grading. This grading is a score which classifies tumors from 1 to 3, where 1 is the least aggressive tumor and 3 is the most undifferentiated tumor. Other clinical differences were distinguished based on the presence of regional node metastases or distant metastases. Most of the tumors were therefore classified within the “TNM” system, where T corresponds to the diameter of the primary tumor, N to the presence of regional nodes, and M to distant metastases. For at least three decades, personalization of oncology was based only on these parameters and on the patient’s physical condition, and even now these represent the fundamental elements for treatment decisions. Chemotherapy, surgery and radiation therapy were once the only treatment options for cancer. Although these treatments are still used, oncologists know that some patients respond better to certain drugs than to others and that a surgical approach is not always indicated. In recent years, researchers have studied thousands upon thousands of samples from all types of tumors. They have discovered that tumors derived from the same body site can differ in very important ways.

Firstly, there is histology*. The pathologist is able to distinguish different subtypes of cancer with the microscope. When a patient is diagnosed with a cancer, he/she will undergo a biopsy or a fine-needle aspiration. In some tumor types, debulking or removal of the primary tumor also allows sampling for tissue examination. Some cells of the tumor which have been removed will be taken and analyzed. This examination allows the pathologist to confirm a cancer diagnosis, but, through particular colorations of the tissue sample, the pathologist is also able to provide clinicians with a lot of additional information, such as the tumor’s histological characterization, its hormone sensitivity, and its grade of differentiation*.

For example, in the treatment of lung cancer the histology provides very useful tools to decide the best drug for the treatment of the patient. Clinical studies have shown that for a patient with lung adenocarcinoma* there might be more chance of a response if the drugs pemetrexed or bevacizumab are added to the chemotherapy, while for a patient with lung cancer of squamous* histology, it would be more beneficial to add gemcitabine or vinorelbine. A similar example may be observed Personalization of Oncological Treatments: The Story 12 for other cancers. For the treatment of esophageal cancer it is mandatory to know if the tumor is squamous or not, because although deriving from the same organ, the treatment approach is completely different.

This information is a useful tool in the first step of the personalization process. For example, lung cancer can be divided as a first step into non-small cell lung cancer and small cell lung cancer, which are two completely different neoplasms*. Within the non-small cell lung cancer category, there are again several different tumor types. Breast cancer can also be divided into two major categories: the hormone-sensitive neoplasms and the HER2-positive diseases. Lung and breast cancers are only two examples, because it is possible to recognize several entities within the same tumor type for many other cancers.

Molecular subsets of lung adenocarcinoma Lung cancer subtypes
Figure 2. Lung Cancer – Not One Disease: Histological (Tissue) and Molecular Subtypes of Lung Cancer (not shown) On the left side, four histological subtypes of lung cancer. On the right side, a pie chart showing the percentage distribution of molecular subsets of lung adenocarcinoma. Adapted from Petersen I. Dtsch Arztebl Int 2011; 108(31-32):525-531 (left) and Pao W & Hutchinson KE. Nature Med 2012; 18(3): 349-351.

Personalization depends on a multidisciplinary approach; we need a range of experts, because we need the medical oncologist, the surgeon and the expertise of the molecular pathologist, who should be part of the team in a more effective, integrated way than before. We don’t need the pathology report alone; we need to interact with all professionals, including nurses, who are dealing with the patient. This, to me, will create a lot of problems in terms of organization of care and in terms of cost, but it is the only way to bring together knowledge on the biology and pathology of tumors for effective treatment in every single patient. Our effort at ESMO is to bring this broad knowledge to the general public, to medical oncologists and to the community of doctors involved in cancer.

We have to deeply analyze each tumor of every patient in order to identify those genetic characteristics that make the tumor able to survive. As a result, we can choose the appropriate drugs to target the specific alterations. The clearest examples of this process are in melanoma, lung cancer and breast cancer. For instance, in lung cancer, the presence of mutations in the epidermal growth factor receptor (EGFR) renders the tumor highly sensitive to EGFR tyrosine kinase inhibitors. When oncologists identify these mutations in a patient’s tumor, they may observe that the lesion disappears a few weeks after treatment. A similar response may be observed after treatment with BRAF inhibitors in patients with melanoma or with gastrointestinal stromal tumors (GIST) that express the c-kit gene. Unfortunately, oncogene addiction is not the only process underlying carcinogenesis* and tumor growth. The tumor environment and so-called “epigenetic” alterations* play an important role in rendering the fight against cancer more and more challenging. Despite the enormous recent advances, a specific alteration has not been identified in all cancers. The hope is that the possibility of sequencing the full genome – which means every gene – will give us new insights and therefore new drugs for our patients.

In the DNA of some individuals a “germline” mutation* may be present. This means that a particular mutation is conferring susceptibility to that person to develop a particular type of cancer during his/her life. For instance, BRCA is an alteration for which there is a particular predisposition to have a breast cancer or ovarian cancer in one’s life. A woman with a BRCA gene mutation can transmit this alteration to her female descendants, so her daughters and following generations of female family members can therefore inherit this predisposition.

Mutations that are not germline are called somatic mutations*, which are acquired mutations and are found generally only in the tumor. Distinct from germline mutations, somatic mutations are not inherited.

The move from blockbuster or empirical medicine* towards personalized medicine is a stepwise process. We are currently on the second step of stratified medicine and moving up the stairs towards personalized medicine.

Will molecular pathology evolve from pathology? You need to give a name to a tumor, and a pathologist is the professional who gives a name to tumors. The variety of cancers is broad; when we say “sarcoma”, “carcinoma”, or “lymphoma”, we actually say nothing, because we have hundreds and hundreds of diseases within these categories that need to be recognized. And the reason for recognizing them is exactly related to personalization. The biology of cancer is very complex, and admittedly we have been very naive in the past. We always thought that the problem was how genes become altered in the cancer cell, but actually it is even more complex than that and also involves the way genes direct how they are read; it is the flow of information that comes from genes to the making of their proteins which is as important as the aberration of the genome.

We are facing obstacles currently because the whole issue of tissue sampling has been regulated under the umbrella of privacy, which is of course important. Defending your rights as a human being is a key issue, but we should also try to focus a little bit on the necessity to use that tissue. Of course, we need to have rules, but the approach we are currently facing is basically preventing clinical research and translational research under the excuse of protecting our privacy as human beings, and this is an increasing obstacle. We as researchers, as molecular geneticists, as pathologists, are really looking into a future in which it is becoming increasingly difficult to try to answer the basic question of cancer genomics. Why? Because it is becoming increasingly difficult to use tissue for these purposes.

With the new therapeutic approach and the use of targeted therapy, molecular testing is gaining a very relevant role. It is very important for us, as advocates, to educate patients in these issues. So patients have to receive very clear and transparent information. It should be the doctor who explains to the patient the reason why molecular testing is performed; the doctor has to explain that molecular testing will find whether there is some tumor characteristic which can be targeted with one of these therapies, in order to determine if maybe the patient is the right candidate to receive targeted therapy and perhaps to benefit from it. The communication between the doctor and patient must be very accurate and must educate, meaning that the patient has to understand the precise situation. This can be important also to empower the patient in treatment decisions, but it is important that he/she knows that not every patient may be a candidate for receiving targeted therapy and to understand why this is the case.

  • Different tumour types are increasingly divided into very small subgroups carrying a rare molecular alteration.
  • Most new drugs are targeting these infrequent events.
  • Clinical trials are testing the use of high throughput molecular technologies* in the context of personalized cancer medicine.
  • There are a growing number of newer techniques to optimize genomic testing, including the virtual cell program, which foresees testing of a piece of patient’s tumor tissue in the laboratory in order to mimic what would happen in the human body (e.g. drug sensitivity).
  • Clinical research is today focusing on target identification at the patient level.

Targeted therapy drugs work differently to standard chemotherapeutic drugs. They attack cancer cells and, in particular, the targets which are strategic points for cell survival, cell replication and metastases. They generally create little damage to normal cells. In fact, these drugs tend to have different side effects to traditional chemotherapeutic drugs. Targeted therapies are used to treat many kinds of tumors: certain types of lung, pancreatic, head and neck, liver, colorectal, breast, melanoma and kidney cancers. Targeted therapies are a major focus of cancer research today

Many future advances in cancer treatment will probably come from this area. There are many different targeted therapies in use and new forms are appearing all the time. Depending on the type of cancer and the way it spreads, targeted therapy can be used to cure the cancer, to slow the cancer’s growth, to kill cancer cells that may have spread to other parts of the body or to relieve symptoms caused by the cancer.

We can divide targeted therapies into two main categories: antibody drugs and small molecules. Antibody drugs are man-made versions of immune system proteins that have been designed to attack the external part of cells at certain targets, generally called receptors. Receptors can be considered the antennas of the cells. They transmit signals from the surrounding environment to the nucleus of the cell. Some receptors are fundamental to the vital processes of the cell. Targeting certain receptors means preventing the transmission of some survival signals to the tumor cells.

Trastuzumab (Herceptin®) is, after tamoxifen, the second targeted therapy drug ever used to treat cancer and it is a monoclonal antibody directed at a receptor called HER2. This targeted therapy greatly improves the survival rate of women with breast cancer expressing the HER2 receptor. Therefore, the determination on tissue blocks of the presence of expression of HER2 is one of the best examples of personalization of treatment.

A knowledge of the cancer characteristics and a determination of the tissue characteristics of each patient allows the doctor to select patients for the best treatment.

Other examples of monoclonal antibodies are cetuximab and panitumumab, which have been developed to treat colon cancer. At first it seemed as if these drugs were a failure, because they did not work in many patients. Then it was discovered that if a cancer cell has a specific genetic mutation, known as KRAS, these drugs will not work.

This is another excellent example of using individual tumor genetics to predict whether or not a treatment will work. In the past, the oncologist would have had to try each therapy on every patient and then change the therapy if the cancer continued to grow.

The other type of targeted therapy drugs are not antibodies. Since antibodies are large molecules, this other type is called “small-molecule” targeted therapy drugs. The small molecules attack cancer cells from the inner vital processes. Also, in this case, the small molecules prevent the broadcast of vital signals that regulate the survival of the tumor. There are several examples of targeted drugs that changed the natural history of some cancers.

One example is imatinib mesylate (Gleevec®), which is used in GIST, a rare cancer of the gastrointestinal tract, and in certain kinds of leukemia. Imatinib targets abnormal proteins, or enzymes, that form on and inside cancer cells and promote uncontrolled tumor growth. Blocking these enzymes inhibits cancer cell growth. Gefitinib (Iressa®) is used to treat advanced non-small cell lung cancer. This drug hits the internal part of the EGFR. These receptors are found on the surface of many normal cells, but certain cancer cells have many more of them. EGFR take in the signal that tells the cell to grow and divide. When gefitinib blocks this signal, it can slow or stop cell growth. However, gefitinib does not work in all patients when trying to treat lung cancer, but only

Personalization of Treatment in a particular subtype. About 10% of patients show genetic alterations called “EGFR mutations” in their tumors at diagnosis. These particular mutations mean that the EGFR is always turned on and therefore there is a continuous signal to the cell to grow and divide. Gefitinib is able to switch off this signal and to stop cell growth in this subtype of patients. After a few weeks, the tumor disappears. Unfortunately, these mutations are rare and they are mainly present in never-smokers, who are the minority of patients.

Another, similar example in lung cancer is provided by crizotinib (Xalkori®). Patients with ALK translocations, which is another rare type of alteration present mainly in never smokers, experience a rapid shrinkage in their tumors when treated with this drug.

Another example of small molecules is represented by sunitinib (Sutent®). This drug is used to treat advanced kidney cancer and some GIST. Sunitinib is considered a multitarget agent because it blocks the vascular endothelial growth factor (VEGF) receptor and other enzymes. By doing all of this, sunitinib slows cancer growth and stops tumors from creating their own blood vessels to help them grow and metastasize. In this case, no biomarkers have been identified to help select patients who are responders from patients who are nonresponders.

Exploring the clinical utility of comprehensive genomic testing. After the patient’s informed consent, tumor and normal DNA is extracted in a certified laboratory. After targeted somatic mutation testing, more extended testing is performed in a research environment. Test results are shared with the treating oncologists, and validation of research findings is pursued if any clinically relevant research findings are found. Therapeutic decisions are based only on validated test results.

We really have to strengthen and reinforce in the future all the collaborative ways to work, without any – or minimal, at least – competitive ways of thinking. We have to work together to make the science evolve and forget about the national or regional representation of research that we have had in the past. I think the priority now is to have really good networks of institutions in order to make new treatments rapidly reach our patients.

3.5 Biomarkers for personalized oncology: recent advances and future challenges.

Kalia M
Metabolism. 2015 Mar;64(3 Suppl 1):S16-21

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells and oncology is a branch of medicine that deals with tumors. The last decade has seen significant advances in the development of biomarkers in oncology that play a critical role in understanding molecular and cellular mechanisms which drive tumor initiation, maintenance and progression. Clinical molecular diagnostics and biomarker discoveries in oncology are advancing rapidly as we begin to understand the complex mechanisms that transform a normal cell into an abnormal one. These discoveries have fueled the development of novel drug targets and new treatment strategies. The standard of care for patients with advanced-stage cancers has shifted away from an empirical treatment strategy based on the clinical-pathological profile to one where a biomarker driven treatment algorithm based on the molecular profile of the tumor is used. Recent advances in multiplex genotyping technologies and high-throughput genomic profiling by next-generation sequencing make possible the rapid and comprehensive analysis of the cancer genome of individual patients even from very little tumor biopsy material. Predictive (diagnostic) biomarkers are helpful in matching targeted therapies with patients and in preventing toxicity of standard (systemic) therapies. Prognostic biomarkers identify somatic germ line mutations, changes in DNA methylation, elevated levels of microRNA (miRNA) and circulating tumor cells (CTC) in blood. Predictive biomarkers using molecular diagnostics are currently in use in clinical practice of personalized oncotherapy for the treatment of five diseases: chronic myeloid leukemia, colon, breast, lung cancer and melanoma and these biomarkers are being used successfully to evaluate benefits that can be achieved through targeted therapy. Examples of these molecularly targeted biomarker therapies are: tyrosine kinase inhibitors in chronic myeloid leukemia and gastrointestinal tumors; anaplastic lymphoma kinase (ALK) inhibitors in lung cancer with EML4-ALk fusion; HER2/neu blockage in HER2/neu-positive breast cancer; and epidermal growth factor receptors (EGFR) inhibition in EGFR-mutated lung cancer. This review presents the current state of our knowledge of biomarkers in five selected cancers: chronic myeloid leukemia, colorectal cancer, breast cancer, non-small cell lung cancer and melanoma.

3.6 Personalized oncology: recent advances and future challenges.

Kalia M1
Metabolism. 2013 Jan;62 Suppl 1:S11-4

Personalized oncology is evidence-based, individualized medicine that delivers the right care to the right cancer patient at the right time and results in measurable improvements in outcomes and a reduction on health care costs. Evolving topics in personalized oncology such as genomic analysis, targeted drugs, cancer therapeutics and molecular diagnostics will be discussed in this review. Biomarkers and molecular individualized medicine are replacing the traditional “one size fits all” medicine. In the next decade the treatment of cancer will move from a reactive to a proactive discipline. The essence of personalized oncology lies in the use of biomarkers. These biomarkers can be from tissue, serum, urine or imaging and must be validated. Personalized oncology based on biomarkers is already having a remarkable impact. Three different types of biomarkers are of particular importance: predictive, prognostic and early response biomarkers. Tools for implementing preemptive medicine based on genetic and molecular diagnostic and interventions will improve cancer prevention. Imaging technologies such as Computed Tomography (CT) and Positron Emitted Tomography (PET) are already influencing the early detection and management of the cancer patient. Future advances in imaging are expected to be in the field of molecular imaging, integrated diagnostics, biology driven interventional radiology and theranostics. Molecular diagnostics identify individual cancer patients who are more likely to respond positively to targeted chemotherapies. Molecular diagnostics include testing for genes, gene expression, proteins and metabolites. The use of companion molecular diagnostics is expected to grow significantly in the future and will be integrated into new cancer therapies a single (bundled) package which will provide greater efficiency, value and cost savings. This approach represents a unique opportunity for integration, increased value in personalized oncology.

3.7  Pharmacogenomic biomarkers for personalized cancer treatment.

Rodríguez-Antona C1Taron M.
J Intern Med. 2015 Feb; 277(2):201-17

Personalized medicine involves the selection of the safest and most effective pharmacological treatment based on the molecular characteristics of the patient. In the case of anticancer drugs, tumor cell alterations can have a great impact on drug activity and, in fact, most biomarkers predicting response originate from these cells. On the other hand, the risk of developing severe toxicity may be related to the genetic background of the patient. Thus, understanding the molecular characteristics of both the tumor and the patient, and establishing their relation with drug outcomes will be critical for the identification of predictive biomarkers and to provide the basis for individualized treatments. This is a complex scenario where multiple genes as well as pathophysiological and environmental factors are important; in addition, tumors exhibit large inter- and intraindividual variability in space and time. Against this background, the huge amounts of biological and genetic data generated by the high-throughput technologies will facilitate pharmacogenomic progress, suggest novel druggable molecules and support the design of future strategies aimed at disease control. Here, we will review the current challenges and opportunities for pharmacogenomic studies in oncology, as well as the clinically established biomarkers. Lung and renal cancer, two areas in which huge progress has been made in the last decade, will be used to illustrate advances in personalized cancer treatment; we will review EGFR mutation as the paradigm of targeted therapies in lung cancer, and discuss the dissection of lung cancer into clinically relevant molecular subsets and novel advances that suggest an important role of single nucleotide polymorphisms in the response to antiangiogenic agents, as well as the challenges that remain in these fields. Finally, we will present new approaches and future prospects for personalizing medicine in oncology.

3.8 Limits to forecasting in personalized medicine: An overview

John Ioannidis
International Journal of Forecasting 2009; 25(4):773-783.

Biomedical research is generating massive amounts of information about potential prognostic factors for health and disease. However, few prognostic factors or systems are robustly validated, and still fewer have made a convincing difference in health outcomes or in prolonging life expectancy. For most diseases and outcomes, a considerable component of the prognostic variance remains unknown, and may remain so for the foreseeable future. I discuss here some of the main problems in medical forecasting that pose obstacles to personalized medicine. Their recognition may help identify solutions to improve personalized prognosis, or at least understand and cope with the component of the future that we cannot predict. Much prognostic research is stuck at generating “publishable units”, without any interest in conclusively proving their worth, let alone moving them into real life applications. Information is reported selectively and reporting is deficient. The replication record of prognostic claims is poor. Even among replicated prognostic effects, few are convincingly shown to add much information besides what is already known through more simple, traditional measurements. There are few efforts to systematize prognostic knowledge. Most prognostic effects are subtle when traced to the molecular level, where most current research operates. Many researchers, clinicians, and the public are not appropriately educated to interpret prognostic information. We still have not even agreed on what the important health outcomes are that we want to predict and intervene for, and some subjectivity may be unavoidable. Finally, without concomitant effective, affordable, and non-harmful interventions, prognosis alone is of questionable value, and wrong prognosis or a wrong interpretation thereof can be harmful. The identification of these problems also suggests a roadmap on what could be done to amend them. Solutions include a systematic approach to the design, conduct, reporting, replication, and clinical translation of prognostic research; as well as the education of researchers, clinicians, and the general public. Finally, we need to recognize that perfect individualized health forecasting is not a realistic target in the foreseeable future, and we have to live with considerable residual uncertainty.
Limits to forecasting in personalized medicine: An overview. Available from: https://www.researchgate.net/publication/223240409_Limits_to_forecasting_in_personalized_medicine_An_overview [accessed May 12, 2015].

3.9 The genome editing toolbox: a spectrum of approaches for targeted modification

Joseph K Cheng,  Hal S Alper

Current Opinion in Biotechnology 2014; 30C:87-94.

The increase in quality, quantity, and complexity of recombinant products heavily drives the need to predictably engineer model and complex (mammalian) cell systems. However, until recently, limited tools offered the ability to precisely manipulate their genomes, thus impeding the full potential of rational cell line development processes. Targeted genome editing can combine the advances in synthetic and systems biology with current cellular hosts to further push productivity and expand the product repertoire. This review highlights recent advances in targeted genome editing techniques, discussing some of their capabilities and limitations and their potential to aid advances in pharmaceutical biotechnology.
The genome editing toolbox: a spectrum of approaches for targeted modification. Available from: https://www.researchgate.net/publication/263816651_The_genome_editing_toolbox_a_spectrum_of_approaches_for_targeted_modification [accessed May 12, 2015].

3.10 The Path to Personalized Medicine

Margaret A. Hamburg, and Francis S. Collins
N Engl J Med Jul 22, 2010; 363(4): 301-304

Researchers have discovered hundreds of genes that harbor variations contributing to human illness, identified genetic variability in patients’ responses to dozens of treatments, and begun to target the molecular causes of some diseases. In addition, scientists are developing and using diagnostic tests based on genetics or other molecular mechanisms to better predict patients’ responses to targeted therapy.

The challenge is to deliver the benefits of this work to patients. As the leaders of the National Institutes of Health (NIH) and the Food and Drug Administration (FDA), we have a shared vision of personalized medicine and the scientific and regulatory structure needed to support its growth. Together, we have been focusing on the best ways to develop new therapies and optimize prescribing by steering patients to the right drug at the right dose at the right time.

We recognize that myriad obstacles must be overcome to achieve these goals. These include scientific challenges, such as determining which genetic markers have the most clinical significance, limiting the off-target effects of gene-based therapies, and conducting clinical studies to identify genetic variants that are correlated with a drug response. There are also policy challenges, such as finding a level of regulation for genetic tests that both protects patients and encourages innovation. To make progress, the NIH and the FDA will invest in advancing translational and regulatory science, better define regulatory pathways for coordinated approval of codeveloped diagnostics and therapeutics, develop risk-based approaches for appropriate review of diagnostics to more accurately assess their validity and clinical utility, and make information about tests readily available.

Moving from concept to clinical use requires basic, translational, and regulatory science. On the basic-science front, studies are identifying many genetic variations underlying the risks of both rare and common diseases. These newly discovered genes, proteins, and pathways can represent powerful new drug targets, but currently there is insufficient evidence of a downstream market to entice the private sector to explore most of them. To fill that void, the NIH and the FDA will develop a more integrated pathway that connects all the steps between the identification of a potential therapeutic target by academic researchers and the approval of a therapy for clinical use. This pathway will include NIH-supported centers where researchers can screen thousands of chemicals to find potential drug candidates, as well as public– private partnerships to help move candidate compounds into commercial development.

The NIH will implement this strategy through such efforts as the Therapeutics for Rare and Neglected Diseases (TRND) program. With an open environment, permitting the involvement of all the world’s top experts on a given disease, the TRND program will enable certain promising compounds to be taken through the preclinical development phase — a time-consuming, high-risk phase that pharmaceutical firms call “the valley of death.” Besides accelerating the development of drugs to treat rare and neglected diseases, the TRND program may also help to identify molecularly distinct subtypes of some common diseases, which may lead to new therapeutic possibilities, either through the development of targeted drugs or the salvaging of abandoned or failed drugs by identifying subgroups of patients likely to benefit from them.

Another important step will be expanding efforts to develop tissue banks containing specimens along with information linking them to clinical outcomes. Such a resource will allow for a much broader assessment of the clinical importance of genetic variation across a range of conditions. For example, the NIH is now supporting genome analysis in participants in the Framingham Heart Study, obtaining biologic specimens from babies enrolled in the National Children’s Study, and performing detailed genetic analysis of 20 types of tumors to improve our understanding of their molecular basis.

As for translational science, the NIH is harnessing the talents and strengths of its Clinical and Translational Sciences Award program, which currently funds 46 centers and has awardees in 26 states, and its Mark O. Hatfield Clinical Research Center (the country’s largest research hospital, in Bethesda, MD) to translate basic research findings into clinical applications. Just as the NIH served as an initial home for human gene therapy, the Hatfield Center can provide specialized diagnostic services for rare and neglected diseases, offer a state-of-the-art manufacturing facility for novel therapies, and pioneer clinical trials of other innovative biologic therapies, such as those using human embryonic stem cells or induced pluripotent stem cells.

Today, about 10% of labels for FDA-approved drugs contain pharmacogenomic information — a substantial increase since the 1990s but hardly the limit of the possibilities for this aspect of personalized medicine.1 There has been an explosion in the number of validated markers but relatively little independent analysis of the validity of the tests used to identify them in biologic specimens.

The success of personalized medicine depends on having accurate diagnostic tests that identify patients who can benefit from targeted therapies. For example, clinicians now commonly use diagnostics to determine which breast tumors overexpress the human epidermal growth factor receptor type 2 (HER2), which is associated with a worse prognosis but also predicts a better response to the medication trastuzumab. A test for HER2 was approved along with the drug (as a “companion diagnostic”) so that clinicians can better target patients’ treatment (see table).

Increasingly, however, the use of therapeutic innovations for a specific patient is contingent on or guided by the results from a diagnostic test that has not been independently reviewed for accuracy and reliability by the FDA. For example, in 2006, the FDA granted approval to rituximab (Rituxan) for use as part of firstline treatment in patients with certain cancers. Since then, a laboratory has marketed a test with the claim that it can identify the approximately 20% of patients who are more likely to have a response to the drug. The FDA has not reviewed the scientific justification for this claim, but health care providers may use the test results to guide therapy. This undermines the approval process that has been established to protect patients, fails to ensure that physicians have accurate information on which to make treatment decisions, and decreases the chances that physicians will adopt a new therapeutic–diagnostic approach. The FDA is coordinating and clarifying the process that manufacturers must follow regarding their claims, including defining the times when a companion diagnostic must be approved or cleared before or concurrently with approval of the therapy. The agency will ensure that claims that a test will improve the care of patients are based on solid evidence, and developers will get straightforward, consistent advice about the standards for review and the best way to demonstrate that the combination works as intended.

In February, the NIH and the FDA announced a new collaboration on regulatory and translational science to accelerate the translation of research into medical products and therapies; this effort includes a joint funding opportunity for regulatory science. Working with academic experts, companies, doctors, patients, and the public, we intend to help make personalized medicine a reality. A recent example of this collaboration is an effort to identify new investigational agents to which certain tumors, identified by their genetic signatures, are responsive. Real progress will come when clinically beneficial new products and approaches are incorporated into clinical practice. As the field advances, we expect to see more efficient clinical trials based on a more thorough understanding of the genetic basis of disease. We also anticipate that some previously failed medications will be recognized as safe and effective and will be approved for subgroups of patients with specific genetic markers.

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