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Posts Tagged ‘cancer treatment’


McEwen Award for Innovation: Irving Weissman, M.D., Stanford School of Medicine, and Hans Clevers, M.D., Ph.D., Hubrecht Institute

Larry H. Bernstein, MD, FCAP, Curator
Leaders in Pharmaceutical Innovation

Series E. 2; 7.3

Past winners include Azim Surani, James Thomson, Rudolf Jaenisch and Kazutoshi Takahashi with Shinya Yamanaka

The International Society for Stem Cell Research (ISSCR) has presented EuroStemCell partner Hans Clevers with the McEwen Award for Innovation at the opening of its annual meeting, today (24 June) in Stockholm, Sweden.

The prizes awarded by ISSCR in 2015 are:

McEwen Award for Innovation: Irving Weissman, M.D., Stanford School of Medicine, and Hans Clevers, M.D., Ph.D., Hubrecht Institute

ISSCR-BD Biosciences Outstanding Young Investigator Award: Paul Tesar, Ph.D., Case Western Reserve University School of Medicine

ISSCR Public Service Award: Alan Trounson, Ph.D., MIMR-PHI Institute of Medical Research

 

In 2015, the ISSCR recognizes long-standing contributors to the field, Weissman and Clevers, for the identification, prospective purification and characterization of somatic (adult) tissue-associated stem cells and advancement of their research findings toward clinical applications.

Award recipient Weissman’s many discoveries have helped map the direction of the stem cell field and have served as the basis for important research and work by scientists all over the world.  He was the first to isolate and characterize hematopoietic (blood) stem cells from mice and humans. He developed the approaches and technologies, now widely used within the field, for isolating blood stem and progenitor cells and defining their properties. Weissman pioneered the extension of his approaches to isolation of other stem cell types, including human nervous system cells and skeletal muscle myogenic stem/progenitor cells. Further, he discovered several independent leukemia stem cells and, more recently, bladder cancer stem cells, head and neck cancer stem cells and malignant melanoma stem cells. Weissman has pursued these discoveries to develop several promising means of cancer therapy.

Award recipient Clevers has been a leader in biomedical sciences and the area of Wnt signaling in colon cancer for more than three decades. He and his lab developed tools to identify and track an adult stem cell population able to give rise to the entire lining of the gut and later to demonstrate that these cells can be isolated and grown in culture as “miniguts,” recapitulating the normal structure and function of the gut. These discoveries are a move toward promising therapies for colon conditions, like ulcers, in which the lining of the intestine has been destroyed in patches, and provide a powerful resource for modeling disease pathology and for drug screening.

“Irv Weissman and Hans Clevers have made enormous contributions to stem cell science. Working in the blood and gut systems, respectively, and extending their findings in different tissues, they have defined the concepts and technologies that underpin many avenues of research,” Hans Schöler, chair of the ISSCR’s McEwen Awards selection committee, said. “Each has made pioneering conceptual advances in disease modeling and regenerative medicine.”

 

The ISSCR-BD Biosciences Outstanding Young Investigator Award recognizes exceptional achievements by an ISSCR member and investigator in the early part of their independent career in stem cell research.  The winner receives a $7,500 USD personal award and is invited to present at the ISSCR’s annual meeting. Past winners include Valentina Greco, Marius Wernig, Cédric Blanpain, Robert Blelloch, Joanna Wysocka and Konrad Hochedlinger.

Award recipient Tesar established his independent laboratory five years ago and has rapidly risen to his current position as the Dr. Donald and Ruth Weber Goodman Professor of Innovative Therapeutics and tenured Associate Professor in the Department of Genetics and Genome Sciences at Case Western Reserve University School of Medicine. Tesar’s studies have shaped the global understanding of both pluripotent stem cell and oligodendrocyte biology. His seminal and highly cited report on epiblast stem cells, published in Nature in 2007, along with similar findings by Pedersen, Vallier and colleagues, led to a complete shift in the understanding of how pluripotency is regulated in the mammalian embryo.  He has continued to provide high impact contributions to the field, pioneering new methods to generate and mature oligodendrocyte progenitor cells, and to use these to enhance repair in animal models of multiple sclerosis.

Stanford stem cell pioneer Irving Weissman wins international honors

by Krista Conger on Feb 10, 2015
http://news.stanford.edu/thedish/2015/02/10/stanford-stem-cell-pioneer-irving-weissman-wins-international-honors/

IRVING WEISSMAN, a professor of pathology and of developmental biology at Stanford Medical School, was recently awarded the Charles Rodolphe Brupbacher Prize for Cancer Research in Zurich.

Weissman, who directs the Stanford Institute for Stem Cell Biology and Regenerative Medicine, was honored for his role in identifying and isolating the first hematopoetic, or blood-forming, stem cell in mice in 1988, and then in humans in 1992. In 2000, he also isolated leukemia cancer stem cells from humans. Recently, he and his colleagues have devoted themselves to understanding how cancer cells escape destruction by the immune system by expressing a “don’t eat me” signal on their cell membranes.

“His discoveries on aging processes in stem-cell systems and ultimately his contribution toward understanding cancer stem cells and the way in which the immune system can control these cells are pioneering achievements with far-reaching clinical implications,” Markus Manz, director of the Department of Hematology at the University Hospital Zurich, said of Weissman at a symposium titled “Breakthroughs in Cancer Research and Therapy” where the prize was announced.

Weissman also is the director of Stanford’s Ludwig Center for Cancer Stem Cell Research and Medicine and holds the Virginia and Daniel K. Ludwig Professorship in Clinical Investigation in Cancer Research.

The prize, presented by the Charles Rodolphe Brupbacher Foundation, included 100,000 Swiss francs, or about $108,000.

The Charles Rodolphe Brupbacher Foundation was founded in 1991 by Brupbacher’s wife, Frederique, in honor of her late husband. This is the 12th time the prize, which is meant to recognize internationally acknowledged achievements in fundamental cancer research, has been awarded. Brupbacher was a Swiss banker, economist and international currency expert.

In addition to the Brupbacher Prize, it was recently announced that Weissman will receive theMcEwen Award for Innovation, supported by the McEwen Centre for Regenerative Medicine in Toronto. The award will be presented in June at the annual meeting of the International Society for Stem Cell Research in Stockholm. It recognizes the work of Weissman and Hans Clevers, of the Hubrecht Institute in the Netherlands, in the identification, purification and characterization of adult stem cells from a variety of human tissues and cancers. Weissman and Clevers will share a $100,000 award.

Anti-CD47 antibody may offer new route to successful cancer vaccination

Scientists at the School of Medicine have shown that their previously identified therapeutic approach to fight cancer via immune cells called macrophages also prompts the disease-fighting killer T cells to attack the cancer.

The research, published online May 20 in the Proceedings of the National Academy of Sciences, demonstrates that the approach may be a promising strategy for creating custom cancer vaccines.

Various researchers have been working over the years to create vaccines against cancer, but the resulting vaccines have not been highly effective. Current approaches to developing the vaccines rely on using immune cells called dendritic cells to introduce cancer protein fragments to T cells — a process known as antigen presentation. The hope has been that the process would stimulate the body’s T cells to identify cancer cells as diseased or damaged and target them for elimination. However, this process often only modestly activates the most potent cancer-fighting kind of T cell, called killer T cells or CD8+ T cells.

The Stanford team discovered that there was another viable vaccine approach, using the macrophage pathway to program killer T cells against cancer. Irving Weissman, MD, professor of pathology and of developmental biology, and his team previously showed that nearly all cancers use the molecule CD47 as a “don’t-eat-me” signal to escape from being eaten and eliminated by macrophages. The researchers found that anti-CD47 antibodies, which can block the “don’t-eat-me” signal and enable macrophages to engulf cancer cells, eliminated or inhibited the growth of various blood cancers and solid tumors.

In the new study, the Stanford team showed that after engulfing the cancer cells, the macrophages presented pieces of the cancer to CD8+ T cells, which, in addition to attacking cancer, are also potent attackers of virally infected or damaged cells. As a result, the CD8+ T cells were activated to attack the cancer cells on their own. “It was completely unexpected that CD8+ T cells would be mobilized when macrophages engulfed the cancer cells in the presence of CD47-blocking antibodies,” said MD/PhD student Diane Tseng, the lead author of the study. Following engulfment of cancer cells, macrophages activate T cells to mobilize their own immune attack against cancer, she said.

The Stanford group plans to start human clinical trials of the anti-CD47 cancer therapy in 2014. The new research provides hope that the therapy will cause the immune system to wage a two-pronged attack on cancer — through both macrophages and T cells. The approach may also give physicians early indicators of how the treatment is working in patients. “Monitoring T-cell parameters in patients receiving anti-CD47 antibody may help us identify the immunological signatures that tell us whether patients are responding to therapy,” said co-author Jens Volkmer, MD, an instructor at the Stanford Institute for Stem Cell Biology and Regenerative Medicine.

The research revives interest in an aspect of macrophages that has been neglected for decades: their role in presenting antigens to T cells. For many years, researchers have focused on the dendritic cell as the main antigen-presenting cell, and have generally believed that macrophages specialize in degrading antigens rather presenting them. This research shows that macrophages can be effective at antigen presentation and are powerful initiators of the CD8+T cell response.

The fact that T cells become involved in fighting cancer as a result of CD47-blocking antibody therapy could have important clinical implications. The antibody might be used as a personalized cancer vaccine allowing T cells to recognize the unique molecular markers on an individual patient’s cancer. “Because T cells are sensitized to attack a patient’s particular cancer, the administration of CD47-blocking antibodies in a sense could act as a personalized vaccination against that cancer,” Tseng added.

Weissman, who is senior author of the new study, is the director of the Stanford Institute for Stem Cell Biology and Regenerative Medicine and the director of the Stanford Ludwig Center for Cancer Stem Cell Research and Medicine.

Other Stanford investigators involved in the research were senior scientist Stephen Willingham, PhD; postdoctoral scholars John Fathman, PhD, Nathaniel Fernhoff, PhD, Matthew Inlay, PhD, and Masanori Miyanishi, MD, PhD; instructor Jun Seita, MD, PhD; graduate student Kipp Weisskopf, MPhil; and life sciences research associate Humberto Contreras-Trujillo.

The research was supported by the Virginia and D.K. Ludwig Fund for Cancer Research, the Joseph and Laurie Lacob Gynecologic/Ovarian Cancer Fund, the National Institutes of Health (grants R01CA86017, P01CA139490, P30CA124435 and F30CA168059), and the Student Training and Research in Tumor Immunology Program of the Cancer Research Institute.

Christopher Vaughan is communications manager at the Stanford Institute for Stem Cell Biology and Regenerative Medicine.

 

Clinical Investigation of a Humanized Anti-CD47 Antibody in Targeting Cancer Stem Cells in Hematologic Malignancies and Solid Tumors

Funding Type:

Disease Team Therapy Development III

Grant Number: DR3-06965

Investigator(s): Irving Weissman – PI

Institution: Stanford University

Disease Focus:
Cancer
Solid Tumor
Blood Cancer

Most normal tissues are maintained by a small number of stem cells that can both self-renew to maintain stem cell numbers, and also give rise to progenitors that make mature cells. We have shown that normal stem cells can accumulate mutations that cause progenitors to self-renew out of control, forming cancer stem cells (CSC). CSC make tumors composed of cancer cells, which are more sensitive to cancer drugs and radiation than the CSC. As a result, some CSC survive therapy, and grow and spread. We sought to find therapies that include all CSC as targets. We found that all cancers and their CSC protect themselves by expressing a ‘don’t eat me’ signal, called CD47, that prevents the innate immune system macrophages from eating and killing them. We have developed a novel therapy (anti-CD47 blocking antibody) that enables macrophages to eliminate both the CSC and the tumors they produce. This anti-CD47 antibody eliminates human cancer stem cells when patient cancers are grown in mice. At the time of funding of this proposal, we will have fulfilled FDA requirements to take this antibody into clinical trials, showing in animal models that the antibody is safe and well-tolerated, and that we can manufacture it to FDA specifications for administration to humans.

Here, we propose the initial clinical investigation of the anti-CD47 antibody with parallel first-in-human Phase 1 clinical trials in patients with either Acute Myelogenous Leukemia (AML) or separately a diversity of solid tumors, who are no longer candidates for conventional therapies or for whom there are no further standard therapies. The primary objectives of our Phase I clinical trials are to assess the safety and tolerability of anti-CD47 antibody. The trials are designed to determine the maximum tolerated dose and optimal dosing regimen of anti-CD47 antibody given to up to 42 patients with AML and up to 70 patients with solid tumors. While patients will be clinically evaluated for halting of disease progression, such clinical responses are rare in Phase I trials due to the advanced illness and small numbers of patients, and because it is not known how to optimally administer the antibody. Subsequent progression to Phase II clinical trials will involve administration of an optimal dosing regimen to larger numbers of patients. These Phase II trials will be critical for evaluating the ability of anti-CD47 antibody to either delay disease progression or cause clinical responses, including complete remission. In addition to its use as a stand-alone therapy, anti-CD47 antibody has shown promise in preclinical cancer models in combination with approved anti-cancer therapeutics to dramatically eradicate disease. Thus, our future clinical plans include testing anti-CD47 antibody in Phase IB studies with currently approved cancer therapeutics that produce partial responses. Ultimately, we hope anti-CD47 antibody therapy will provide durable clinical responses in the absence of significant toxicity.

New insights into the biology of cancer have provided a potential explanation for the challenge of treating cancer. An increasing number of scientific studies suggest that cancer is initiated and maintained by a small number of cancer stem cells that are relatively resistant to current treatment approaches. Cancer stem cells have the unique properties of continuous propagation, and the ability to give rise to all cell types found in that particular cancer. Such cells are proposed to persist in tumors as a distinct population, and because of their increased ability to survive existing anti-cancer therapies, they regenerate the tumor and cause relapse and metastasis. Cancer stem cells and their progeny produce a cell surface ‘invisibility cloak’ called CD47, a ‘don’t eat me signal’ for cells of the native immune system to counterbalance ‘eat me’ signals which appear during cancer development. Our anti-CD47 antibody counters the ‘cloak’, enabling the patient’s natural immune system to eliminate the cancer stem cells and cancer cells. Our preclinical data provide compelling support that anti-CD47 antibody might be a treatment strategy for many different cancer types, including breast, bladder, colon, ovarian, glioblastoma, leiomyosarcoma, squamous cell carcinoma, multiple myeloma, lymphoma, and acute myelogenous leukemia.

Development of specific therapies that target all cancer stem cells is necessary to achieve improved outcomes, especially for sufferers of metastatic disease. We hope our clinical trials proposed in this grant will indicate that anti-CD47 antibody is a safe and highly effective anti-ancer therapy that offers patients in California and throughout the world the possibility of increased survival and even complete cure.

We have previously developed a new therapeutic candidate, the anti-CD47 humanized antibody, Hu5F9-G4, which demonstrates potent anti-cancer activity in animal models of malignancy. The goal of CIRM DTIII Grant DR3-06965 is to conduct initial phase I clinical trials of this antibody in advanced cancer patients. We originally proposed to conduct two separate Phase I clinical trials: one in solid tumor patients with advanced malignancy (commenced in August 2014), the other in relapsed, refractory AML patients (anticipated to start in September 2015). The primary endpoints for these trials will be to assess safety and tolerability, and additional endpoints include obtaining information about the dosing regimen for subsequent clinical investigations, and initial efficacy assessments.

CD47 is a dominant anti-phagocytosis signal that is expressed on all types of human cancers assessed thus far. It binds to SIRPα, an inhibitory receptor on macrophages, and in so doing, blocks the ability of macrophages to engulf and eliminate cancer cells. Hu5F9-G4 blocks binding of CD47 to SIRPα, and restores the ability of macrophages to engulf or phagocytose cancer cells. In pre-clinical cancer models, treatment with Hu5F9-G4 shrunk tumors, eliminated metastases, and in some cases resulted in long-term protection from cancer recurrence. These results suggest that Hu5F9-G4 leads to elimination of cancer stem cells in addition to differentiated cancer cells.

We have developed Hu5F9-G4 for human clinical trials by demonstrating safety and tolerability in pre-clinical toxicology studies. These studies also indicated that we can achieve serum levels associated with potent efficacy in pre-clinical models. The regulatory agencies (FDA in the U.S., and MHRA in the U.K.) reviewed the large package of pre-clinical data describing Hu5F9-G4, and approved our requests to commence separate Phase I clinical trials in solid tumor and AML patients. The solid tumor trial commenced at Stanford in August 2014 and has been designed to assess patients in separate groups, or cohorts, treated with increasing doses of Hu5F9-G4. The trial is ongoing as primary endpoints have not been met. The acute myeloid leukemia trial has been given regulatory approval in the U.K., and will start enrolling patients in September 2015. In summary, during the last year, the Hu5F9-G4 clinical trials have made substantial progress and all milestones have been met.

Stem Cell Research: Promise and Progress

Hans Clevers: “Every day new research is showing us that many types of cancers are fed by tumour stem cells”

http://www.irbbarcelona.org/en/news/hans-clevers-every-day-new-research-is-showing-us-that-many-types-of-cancers-are-fed-by-tumour

The biggest challenge in designing new cancer therapies lies in successfully identifying and targeting tumour stem cells, which are responsible for the regrowth of the tumour.

The Barcelona BioMed Conference on “Normal and Tumour Stem Cells”, aims to analyze the function of stem cells in cancer. The conference, which begins today and runs until November 14 at the Institut d’Estudis Catalans, is co-organized by colon cancer research experts Eduard Batlle (IRB Barcelona) andHans Clevers (Hubrecht Institute, the Netherlands), with the support of the BBVA Foundation. During the three-day event, 21 world experts in the field will meet with a further 130 participants to share their latest research findings on tumour stem cells.

“In 2007 we held the first Barcelona BioMed Conference on this topic. At the time there was only very preliminary data on the relationship between stem cells and cancer. Five years on, many convincing data have emerged to indicate that the majority of tumours are indeed fed by tumour stem cells,” explains Hans Clevers, the scientist who first identified stem cells in the intestine and who today is one of the world leaders in research on normal stem cells and their potential for regenerative therapy.

A number of important studies have demonstrated that at the heart of cancers of the breast, colon, skin, brain, lung and leukemias lie a small group of malignant cells that have retained the properties of the stem cell that gave rise to the cancers in the first place. It is these cells that allow the tumour to grow and can regenerate it. The efforts of many research groups worldwide now focusses on unraveling this process, identifying the specific genes that allow it to occur, and finding ways to detect and eliminate these malignant stem cells.

Stem cells and the origin of tumours

One of the principal characteristics of stem cells is that they are able to copy themselves indefinitely, giving rise to one stem cell and one specialized cell. This capacity for unlimited replication ensures the constant renewal of healthy tissues, which is fundamental for survival and is the basis of regenerative medicine. When the stem cells undergo cancerous mutations or when normal tumour cells acquire stem cell properties, however, this can lead to the formation of tumours.

“This conference gives us a valuable opportunity to learn about the latest work on the two types of stem cells, normal and tumour, in different tissues. What we have been observing over recent years is that the tumour mimcs the hierarchies that exist in normal tissues. In order to understand the tumour, we need to understand the healthy tissue. Most of the scientists invited to the conference are working on both aspects,” explains Batlle. The list of speakers includes pioneers in the field, such as Irving L. Weissman, director of the Institute for Stem Cell Biology & Regenerative Medicine in Stanford, California. Weissman, known as the “father of haematopoiesis”, first identified stem cells in the blood and determined how they give rise to the different types of blood cells, making major contributions to our understanding of leukemias and other ‘liquid’ tumours.

Stem cells and metastasis

In addition to being at the root of the tumour and allowing it to grow, stem cells may also cause metastasis. In order for metastasis to occur, cells from the original tumour must escape into the blood stream and invade new organs to seed new tumours there. “Only cells with stem cell properties are able to make this happen, since they are the only type of cell that can generate all the cell types of the tumor,” explains Batlle. But in order to cause metastasis, these cells also need to be able to do other things. “We have discovered that in the case of colon cancer, stem cells must be able to trick the healthy tissue of the organ they have invaded into helping them survive in this hostile environment.” Batlle’s study, to be published tomorrow inCancer Cell, will be presented during the conference. This is the first piece of work to reveal a key role for the tumour microenvironment in fostering the process of metastasis, a discovery which will open doors to similar findings in other types of tumours.

Normal stem cells vs. tumour stem cells

One of the keys in the fight against cancer is the ability to identify tumour stem cells and differentiate them from healthy stem cells. The conference co-organizers maintain that “this is still a central question. We don’t yet know enough about normal stem cells, and technical issues make things difficult. We are making rapid progress, however, and in the next few years we expect to be able to make great strides both in figuring out the similarities and differences in the two types of cells, and in coming up with new strategies to fight the growth and spread of tumours.”

PROFILES OF CONFERENCE CO-ORGANIZERS

EDUARD BATLLE – Group Leader of the Colorectal Cancer Laboratory and Coordinator of the Oncology Programme at IRB Barcelona. ICREA Research Professor (Instituto Catalán para la Investigación y Estudios Avanzados).

Dr. Batlle’s research over the past decade has focused on the characterization of the mechanisms that cause the initiation, progression and metastasis of colon cancer. He has published studies in several high-impact journals such as Cell, Nature, Nature Genetics and Cancer Cell. His achievements include the discovery of the transcription factor Snail in tumour cells and the elucidation of the function of EphB membrane receptors in colorrectal cancer. During the Barcelona BioMed Conference, Dr. Batlle will present the results of a study to be published in Cancer Cell on a process indispensable for colon cancer metastasis.

Among his recognitions, Batlle has received the Banc Sabadell Prize for Biomedical Research (2010) and the “Debiopharm Life Sciences Award for Outstanding Research in Oncology” given by the Ecole Polytechnique Fédérale de Lausanne in Switzerland (2006). He is the recipient of an ERC Starting Grant awarded by the European Research Council in 2007.

 

HANS CLEVERS – Group leader at the Hubrecht Institute (director 2002-2012 ) and President of the Royal Netherlands Academy of Arts and Sciences. Dr. Clevers was the first scientist to identify intestinal stem cells and remains one of the leading researchers in this field. His discoveries have had significant impact in cancer as well as in regenerative therapy with stem cells and in vitro organ culture. Clevers’ work in developmental biology and cancer led him to discover the beta-catenin/Tcf4 transcriptional complex, which causes the majority of colorrectal cancer.

http://apoorvamandavilli.com/wp-content/uploads/2010/10/2010stem-cells-and-cancer.pdf

 

In 1991 Clevers became a professor of immunology at the University Medical Center in Utrecht. Since 2002 he has been a professor of molecular genetics at UMC Utrecht. Also in 2002 he became director of the Hubrecht Institute for Developmental Biology and Stem-Cell Research at the Royal Dutch Academy of Sciences, where until May 2012 he led the WNT Signaling and Cancer research group and was project leader of the Netherlands Proteomics Centre and Cancer Genomics Centre. Clevers discovered similarities between the normal renewal of intestinal tissue and the onset of colon cancer. In 2007 he received a grant of two million euros from the KWF Cancer Society to study the function of stem cells in the normal intestines and in colon cancer, and in 2008 he received an ERC Advanced Investigator Grant. In March 2012, Clevers, who since 2000 had been a member of the Royal Netherlands Academy of Arts and Sciences, was elected its president, a position he assumed on June 1 of that year, succeeding Robbert Dijkgraaf. In connection with his election to this position, he resigned from the Hubrecht Institute and began to carry out research two days a week at the UMC-U.[4][5][6][7][9]

Asked in a 2008 interview what had been the highlights of his research up to that point, Clevers said “there would probably be three. There was a first one, when I just started my lab, within the first few months we cloned the gene that they call TCF1, t-cell factor 1, I used to be a t-cell embryologist when we first started out. And that paper was published in EMBO in ’91, first author. So in that paper we described cloning of this vector, which at that time maybe on the world scale was not great but for my own lab to clone this gene was my first thing I ever did alone. This gene then in ’96 we found to be the crucial missing component of what’s called the Wnt signaling pathway, and this [was] generally seen as a major breakthrough we had. There were papers in ’96 and ’97 in Cell, and we had two papers in Science in the same two years.”

Clevers and his team thus showed that “there is that this TCF transcription factor, there is a small family of them, they occur in every animal on the planet, they are the end point of the signal transcription cascade, and they control virtually every decision in a developing animal. When we realized this we started changing our model systems, we used to work on lymphocytes, and we changed it, first to frogs and flies, drosophila, where the Wnt pathway had been studied by many other people that way we could use assays of those people. We then realized that in mammals Wnt signaling…was not only important in embryos but also crucial in adults, which is novel. And we switched to the gut, we found that one of our knockouts, the TCF4 knockout, one of the four members of that family had no stem cells in the gut. And this is the first link in the literature, this was also a ’97 paper in Nature Genetics, between Wnt signaling and stem cells in adults. And in that same year we found that colon cancer comes about by the disregulation of TCF4, and those two phenomena are really linked. So stem cells need TCF4, cancers disregulate TCF4 by mutating a gene upstream in that pathway called APC.”

After this Clevers’s team “continued to work on the intestine and on the physiology of the intestine, which was essentially an unstudied field, much to my surprise. May I emphasize, there are thousands of very competent embryologists, and they work on tiny details, and they fight over the smallest details, are extremely competent. In this intestinal field there are thousands of gastroentromologists that study cancer or colitis or Crohn’s Disease, but there are very few, if any, labs studying normal tissue, which is amazing because that is a tissue that we use every five days. It’s the most rapidly proliferating tissue in a normal body. So my lab actually build up a lot of mouse models and we learn a lot about how that’s being done, and then finally…last year we finally identified the stem cells in the gut. And we now can purify them in large numbers and study their characteristics.”[4]

A recent posting at the website of the Royal Netherlands Academy of Arts and Sciences provides a capsule summary of Clevers’s research to date: “His research deals with the intestine, in both its healthy and diseased state. He has discovered that there are numerous similarities between the normal process whereby intestinal tissue is renewed and the development of intestinal cancer. Improved understanding of these processes is crucial to developing new ways of treating cancer. Hans Clevers has described the molecular signalling pathways that are disrupted by cancer and has identified a protein that is specific to stem cells in the intestine. He has then been able to grow ‘mini-intestines’ from individual stem cells. These are the first steps on the road to regenerative medicine, in this case the regeneration of intestinal tissue.”[7]

Q&A: Hans Clevers

Eric Bender

Nature 521, S15 (14 May 2015) http://dx.doi.org://10.1038/521S15a

n 2009, Hans Clevers and Toshiro Sato (then a postdoc in Clevers’ lab) demonstrated a powerful new model to study development and disease: a three-dimensional ‘organoid’ derived from adult stem cells that replicates the structure of cells lining the intestine. More than 100 labs worldwide are now working with different types of organoid to study cancer and other diseases. Clevers, at the Hubrecht Institute in Utrecht, the Netherlands, discusses the potential of this approach.

Why might it be better to screen drugs in organoids rather than in cell lines?

We don’t currently understand why certain tumours are sensitive or resistant to particular drugs. With targeted therapies, you can make a prediction, but for classical chemotherapy drugs, such as cisplatin or 5-fluorouracil, it is totally unpredictable which tumours will respond. Tumours can be sequenced in great detail, but drugs against them cannot be tested effectively other than in clinical trials. Organoids are a very good genetic representation of the tumour, so they let us bridge the gap between deep-sequencing efforts and patient outcomes.

How do you see organoids contributing to the study of colorectal cancer?

We are collaborating with groups at the Broad Institute in Cambridge, Massachusetts, and the Sanger Institute in Hinxton, UK, to build a biobank of organoids from 20 or so people with colon cancer. We have organoids of the cancer and of normal cells from individual patients, as well as sequences of their protein-coding genes. We have established the non-profit Hubrecht Organoid Technology (HUB) to expand our organoid biobanks. The HUB shares these biobanks with academic groups around the world, and now works with about 15 companies on drug-development programmes. We can culture tumours from almost every person with colon cancer, sequence them and test them against drugs. Additionally, we can use research techniques that have been developed for cell lines, such as genetic tools, fluorescence-activated cell sorting and microarrays.

Is this research moving towards clinical trials?

Yes, my group and the HUB are collaborating with Emile Voest at the Netherlands Cancer Institute in Amsterdam on an observational trial. We already have some organoid models from people with colon cancer who receive chemotherapy. The organoids are screened against a panel of common colon-cancer drugs. The patients will be treated the same way the oncologists would normally treat them, but we’ll see if we could have predicted the response from our organoids. We’re also starting another trial in which we will enrol advanced-colon-cancer patients, for whom there is no standard treatment. We will make organoids, test drug sensitivity and resistance, and then advise the oncologists as to what drug to use for that particular patient. We will be looking at multiple drugs, so we need large numbers of patients — that’s the only way we will be able to produce enough data to help us match drugs to tumour types.

To benefit individual patients, won’t you need to test the drugs very quickly?

Yes — and that’s really where we want to take this technology. When you have pneumonia, your bacterial cultures are tested and you get answers in three days. With this technology, we can tell the oncologist the best odds for a combination of therapeutics, maybe not in three days, but in several weeks. We have an organoid-based test in cystic fibrosis that gives us a result in about two weeks.

How does the organoid approach differ from patient-derived xenografts, in which patients’ tumours are transplanted into immune-suppressed mice for testing drugs?

It’s the same principle — you get a functional readout of the patient’s tumour. But organoids can be tested against an unlimited amount of compounds and combinations. Furthermore, in contrast to xenografts, organoids can be established from almost all patients.

What are some of the next steps in your cancer research?

Organoids model the key component of the tumour but they lack some important elements. We want to combine organoids with other elements to make more-complete tools. For instance, we would like to introduce the immune system so that we can study the effects of the fantastic new immunotherapy drugs. We think that we can build it up in a reductionist way — take lymphocytes isolated from a tumour, bring these together with cancer organoids derived from the same tumour and watch what happens. And maybe we can also put microorganisms in these organoids. For example, we could add Helicobacter, a major cause of stomach cancer, to stomach organoids.

Can organoids also help to test drug combinations?

Yes, tumours are genetically heterogeneous, and there can be vast differences in drug sensitivity between clones for the same tumour. We can possibly advance sequence-based therapy by testing millions of drug combinations in organoids.

Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche

Toshiro Sato1, Robert G. Vries1, Hugo J. Snippert1, Marc van de Wetering1, Nick Barker1, Daniel E. Stange1, Johan H. van Es1, Arie Abo2, Pekka Kujala3, Peter J. Peters3 & Hans Clevers1
Nature 459, 262-265 (14 May 2009) |   http://dx.doi.org:/10.1038/nature07935    Received 16 July 2008; Accepted 24 February 2009

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5+ stem cells at the bottoms of small-intestinal crypts1. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ stem cells can also initiate these crypt–villus organoids. Tracing experiments indicate that the Lgr5+ stem-cell hierarchy is maintained in organoids. We conclude that intestinal crypt–villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.

  • A Model for Life
Dis. Model. Mech. September 2013, doi: 10.1242/dmm.013367 vol. 6 no. 5 1053-1056

A gutsy approach to stem cells and signalling: an interview with Hans Clevers

Hans Clevers, Professor of Molecular Genetics at Utrecht University, began his career in immunology and developmental biology, but a shift towards intestinal research in the late 1990s led to his group’s pioneering discovery that Lgr5 is a marker of tissue stem cells – a finding that paved the way for a cascade of key insights into the molecular signalling pathways that are dysregulated in cancer. Interviewed here by Ross Cagan, Editor-in-Chief of Disease Models & Mechanisms, Hans recalls the mentors and discoveries that motivated his transition from basic to applied science, discusses his style of lab management and mentorship, and highlights the potential of organoid-based therapy for personalised medicine.

Johannes (Hans) Clevers was born in 1957 in Eindhoven, home to Philips Electronics, in the south of The Netherlands. From a young age he showed enthusiasm and a natural talent for science, and as an undergraduate became fascinated with molecular biology. He obtained his PhD in immunology from Utrecht University during the mid-1980s, and simultaneously studied medicine. Making the pivotal decision to move back into the lab after completing his clinical training, he undertook postdoctoral research in Cox Terhorst’s lab at the Dana-Farber Cancer Institute at Harvard University. He then returned to Utrecht to set up his own lab, and was a Professor of Immunology at the university between 1991 and 2002. From 2002 to 2012 he was Director of the nearby Hubrecht Institute for Stem Cell Research. During this time, Hans moved gradually into the gastroenterology field, and made groundbreaking discoveries regarding the role of Wnt signalling in stem cells and colon cancer. His unique contributions to cancer, stem cell research and regenerative medicine have been recognised in the form of numerous awards, and in 2013 he was one of the eleven winners of a $3 million award from the Breakthrough Prize in Life Sciences Foundation. Currently, he is Professor of Molecular Genetics at Utrecht University, and is also President of the Royal Netherlands Academy of Arts and Sciences (KNAW). Hans has also been involved in setting up several biotechnology companies.

Before we get to your background, I want to congratulate you on being, unsurprisingly, one of the Breakthrough Prize award winners. You have a long list of prizes now – is it something you’ve gotten used to?

This last one was unusual for me – prior to the Breakthrough award I had only ever received one American prize and that was in gastroenterology. To be the only researcher in Europe awarded, and to see my name on the list together with people like Robert Weinberg and Bert Vogelstein, who were the big shots when I was a postdoc, was a truly great honour. I went to the ceremony for the physics prize in Geneva, and it was like being at the Oscars – very surreal, as a scientist.

The first thing I did when I found out about my award was to invite the current and previous members of my lab to a huge party in Amsterdam, which will take place in September [2013]. There will be around 100 attendees – most of which are still in science. There will be good food and drink, stand-up comedy, and a small symposium.

Taking a step back into your past, why did you choose a career in science and medicine?

My high school system was very geared towards languages. I started learning biology at university in 1975 at the age of 18, and I was disappointed. Molecular biology was being developed in England, Switzerland and the US, but in Dutch universities there was no legal framework to do this, and so the courses – where available – focused only on technical details. Biology in general lacked charisma. At the time, my friends and brothers were junior medics, and as I had an interest in medicine I decided to take it on in addition to biology. I ended up spending a year in Nairobi and half a year at NIH for my biology rotations, and essentially I never went to any lectures (although this is something I never tell my students!). Anyway, I really started getting sucked into the clinical training, and found that working in a clinical environment is much more sociable than being in a lab. You’re part of a big organisation and there are lots of people to talk to, whereas in the lab there are only a few people, and small issues – such as somebody not cleaning up – can really cause friction. After medical school, I was picked, mainly because of my research background, for a training position in paediatrics. They suggested that I should start work for a PhD, so I went back into the lab. That’s when I realised that, despite the social attractiveness of working in a hospital, I was much more of a scientist than a doctor. I got my PhD – together with four published papers – in just 1 year. However, it was during my first postdoc position in Boston that I think I was really exposed to science for the first time. It was tough, but I knew I’d made the right decision.

Are there particular mentors who influenced your decision to choose the lab over clinics, and shaped your career moves?

When I received the Heineken Prize from the Royal Netherlands Academy of Arts and Sciences in 2012, I had to think deeply about my mentors and realised that there were two that I had almost forgotten. The first was my high school chemistry teacher, who sold laboratory chemicals to students from his home, during the evenings (in a well-regulated way). I had built a small lab in the attic of my parents’ house and I really had fun mixing things together and doing all the experiments that are possible to do at home. Because of this chemistry teacher, I learned the joy of being in a lab.

The second crucial mentor was my thesis advisor, who didn’t supervise me very much but did give me key advice that has stayed with me until now. He taught me that it’s important to trust everybody you work with, at least until they show you that they can’t be trusted. I emphasize this in my own lab – I encourage my students and postdocs to be open and transparent and to discuss their work. Some scientists are intuitively secretive and paranoid – cultural differences perhaps play a part in this. In my view, only when someone damages your trust can you justify being paranoid, and until then it is important to share information.

“…it’s important to trust everybody you work with, at least until they show you that they can’t be trusted”

There are many ways to run a lab; for example, you can micro-manage it or you can focus on the big picture and step back from the day-to-day issues. What is your style of running a lab?

When I first became a PI, I really liked doing experimental work. Even after 5 years as a postdoc, I enjoyed doing minipreps! As a consequence, I really micro-managed the few lab members I had, and I’m sure they were ultimately happy to get away from me. But when the lab grew a little bigger and I became Head of Department, it took me away from the lab much of the time. Nowadays, I informally talk with my lab colleagues as much as I can, preferably at the bench. As we speak, I know that there is someone in my group who will find out the results of a 3-month effort, today. I always insist on looking at the raw data, never the digested, analysed data. It could be 5 minutes or 2 hours, but when I’m needed in the lab I will always try to make time for it and be part of the troubleshooting process. When you can no longer troubleshoot in your own lab, you’re lost.

Well clearly success builds on success – some impressive scientists have come out of your lab. Do you encourage all of your group members to pursue academic positions?

I’ve had many ‘super postdocs’ in my lab but some of these individuals would not be happy as PIs. It’s not about capability, but about wanting to deal with the paperwork, the responsibility and the decision-making that come with being a PI. Such individuals can make a valuable contribution to a lab, given their years of experience, as well as acting as great mentors and role models for the newer group members. When, having gained experience in the pharmaceutical industry, Nick Barker re-joined my group in 2006 as Senior Staff Scientist, we spent 6–7 years looking for stem cell markers, and then broke open the field by identifying Lgr5 as a marker of cancer stem cell populations. Nick has now set up his own group in Singapore, but I have had several other very talented experimentalists in my lab for many years. Overall, I think that intermediate positions are fantastic for successful postdocs who might end up unhappy as PIs.

How did you get involved with intestinal stem cell research? You didn’t start in this field but somehow ended up there.

As an undergraduate student, I did a brief rotation project on T cells. This led to a PhD and postdoc focused on T cells. I learned molecular biology, which inspired me to clone a T-lymphocyte transcription factor, TCF-1, when I subsequently set up my own lab in Holland. We (Marc van der Wetering and I) cloned TCF-1 within a few months and showed that it binds DNA; but, despite trying all kinds of functional assays, we couldn’t show that it regulates transcription. It took 6 or 7 years to figure out that β-catenin, a signal transducer in the Wnt signalling pathway, was needed. We heard that Walter Birchmeier had made a complementary discovery in Berlin, and our papers came out at the same time.

Around that time, I was Clinical Professor in Immunology at Utrecht, and I started studying TCFs in mice, frogs, flies and worms. We soon established that TCFs are always the endpoint of the Wnt pathway. In 1996–1997, we knocked out TCF-4 in mice and, remarkably, observed a gut phenotype – the mice had no crypts. Simultaneously, we realised that the pathway is overactivated in colon cancer. That’s when I decided to move into studying the gut. It wasn’t easy as an immunologist, but I gradually got to know the gastroenterology field. At the time, this field was dominated by clinical research, and in fact our work didn’t really become known to gastroenterologists until around 3–4 years ago. They were totally unaware that mice could give clues about human disease, which surprised me, as in haematology and immunology, there is a good balance between basic and clinical science. There are other clinically well-developed fields, such as prostate and lung cancer research, that could really benefit from a stronger basic approach.

A key discovery for you was that Lgr5 is a marker of stem cells. When did you realise the implications of this discovery?

There were two ‘eureka’ moments with the stem cell story. The dogma at the time was the ‘+4’ stem cell model, which was pioneered by Chris Potten, who recently passed away. I tried to provide experimental support for this model, together with Nick Barker, but it never really went anywhere. Having realised that β-catenin and TCFs controlled crypts in the gut and cancer, we set out to determine the genetic programme controlled by this pathway. At the time (1997), there was no technology to do this properly, but in 2000 we performed one of the first microarrays with Pat Brown. Our array looked at expression in a colon cancer cell line. The array contained only two samples – plus or minus the Wnt pathway – but it opened the field for us by providing a list of markers to investigate further. This was the first, key step. From the list of markers, we picked a few that we thought were marking +4 cells, but these led us nowhere. Eventually, based on its unique expression pattern, we came up with Lgr5. We made numerous mouse strains, including Lgr5-GFP tagged mice. The moment we saw tiny cells lighting up under the microscope, I started writing our next ten big papers in my head. It was a remarkable moment – the cells exist, and we could visualise them using these mice.

And why exactly is Lgr5 so important, both from a basic and an applied standpoint?

Lgr5 is an exquisite protein. We and several other labs have shown that it is a marker for stem cells in many tissues. Originally, we saw it only in spontaneously dividing tissues, but we’ve recently found that it also appears in organs that have undergone damage. Lgr5 is unique in that it – on its own – it specifically marks homogenous populations of stem cells but not their progenitors, unlike most other markers. We now know that this is because it is a cell surface receptor protein in the Wnt pathway, and only stem cells require Wnts. In the gut, the stem cells are particularly active – in mice, they divide every day for 2.5 years, so they go through a thousand cell divisions.

Discovering Lgr5 led to another eureka moment: the generation of long-term culture systems that maintain crypt physiology. A Japanese gastroenterologist who I invited to my lab, Toshiro Sato, was the first to set up the right culture conditions, and now multiple labs are creating these systems, which are called organoids or ‘mini-guts’. Once the system was up and running, Toshiro showed that Paneth cells provide the niche for stem cells at crypt bottoms, and that stem cells produce their own daughters which then produce growth factors. With his former Japanese lab, we showed that normal tissue can be generated from a single stem cell, and it can survive in a mouse for as long as you want. Based on this finding, our lab evolved and now we’re culturing prostate, liver, pancreas, kidney, lung and breast tissue, all for prolonged periods of time, all from humans. There are no changes in chromosomal structure in the cultured cells, and deep sequencing reveals very few mutations. The next step will be to take single cells, genetically modify them like we do with embryonic stem cells, pick a safe clone, expand it and use it for therapy, particularly transplantation.

Do you think we will be able to take organoid-based therapy to the personalised level? Colorectal cancer, for example, only has a 3% success rate in clinical trials. Are organoids going to provide the answer?

We’re finalising a pilot sequencing study now involving 20 patients with normal crypts and colon cancer. With the wild-type and colon cancer organoids, we can potentially predict patient outcome and response to drugs. In the future, we hope to rapidly build large, living biobanks for other cancers, too. In line with this, we’re building up a ‘Stand Up 2 Cancer’ dream team involving several American labs and the Sanger Institute, with the aim of taking the organoid approach to the next level in cancer therapy. Sanger has robotised screening set-ups that allow thousands of compounds to be screened across hundreds of cell lines. We can now do this with patient-derived organoids. From these tests we could establish new effective drug combinations, and we could link genetics to function to help design smarter trials. The great thing about organoids is that they contain only epithelium – there is no immune system, no blood system, only the diseased tissue, making it a very clean system.

We’ve also recently collaborated with clinicians on a cystic fibrosis project. We can predict using cystic fibrosis ‘mini-guts’ that certain drugs that are currently in trials will work for one patient and not for another, and that certain drug combinations work better than others. From biopsy to drug response, it takes only 10 days. Industry is now very interested in using this assay to pre-screen and design trials.

“The great thing about organoids is that they contain only epithelium – there is no immune system, no blood system, only the diseased tissue, making it a very clean system”

In the past, you’ve suggested that classic hypothesis-driven science isn’t the right way to do science. Could you say a little bit more about this?

Now that I’m a bit older I’m more interested in how the process of science works. I always ask my colleagues: how do you run the lab and how do you make discoveries? In my lab, I try to establish a reproducible, quantitative system, like GFP mice and arrays. Then, I throw something at the system and look, without formulating a hypothesis. This is difficult because our brains like to produce causal relationships, even though these are often wrong. I’m constantly telling my group members that they should keep their minds open and make observations without assuming that they know what’s going on. In molecular biology, we can go anywhere we want and there are billions of effects to discover. You cannot do this in a hypothesis-driven way because you’re essentially retracing evolution. There are many solutions to a particular problem but evolution picked one – it’s very arrogant to think we can reconstruct this in our minds.

Some of my most elegant hypotheses have fallen by the wayside. The importance of establishing formal rules for innovation is a discussion worth having in biology. I understand that you have embraced movies to explain scientific concepts. What’s the story behind this?

I was inspired by Leonard Zon – I came across one of his movies about 8 years ago. I realised it’s much easier to convey messages visually than in words so I started working with a small company in Holland to produce science movies. The lab provides the idea and the images, and the company writes the script. We end up going back and forth a few times to make the message as accurate as possible, and it really shows us as scientists how ambiguous language can be. Often, feedback from the company sends us back into the lab to find out something we hadn’t looked into, for example how fast do the cells move, how many cells are there? Gradually, the movie comes together. Nowadays, I typically use the movies in my talks to explain a problem, and I’ve found that it’s much more effective to show the movie before explaining the experiments. People understand the experiments much better that way, and listen effortlessly. Now, whenever we have a story to write up I try to turn it into a 30-second movie before putting pen to paper. This really forces us to think about the core of the paper.

“In molecular biology, we can go anywhere we want and there are billions of effects to discover…There are many solutions to a particular problem but evolution picked one – it’s very arrogant to think we can reconstruct this in our minds”

In your view, is being a scientist a good career choice? What advice would you give to a young scientist thinking about this career?

Science is frustrating because things don’t work 90% of the time: ideas are wrong, experiments fail. You have to have the personality that thrives by those few fantastic moments of success that you have once a year or even once a career. Moving from being a clinician to being a scientist was one of the hardest decisions I ever made. A clinician gets rewards multiple times a day, so if you’re a person who needs that kind of reward and social interaction, then you shouldn’t be a scientist. Luckily there are now many alternative careers, such as pharma, government and teaching, that didn’t exist when I was a young scientist. However, there needs to be a radical change in the way we view these alternative routes. Maybe in the US it’s different, but here, if you step out of the system you are treated like a failure. I tell young scientists that failure comes with ending up as a miserable PI, with no funding and no papers.

PhD students and junior postdocs have to be aware that the people they see at meetings who give the great talks are in the minority – as scientists we have to be ready to do something else at any point during our career. I think the whole system has to realise that every other job can be as interesting as a job in science. That’s not what we always convey to young people – we describe academia as where it’s happening and everything else as dull or uncreative.

If you hadn’t chosen science as a career, what would you have done instead?

I would probably be a novelist. It’s even more competitive than being a scientist, but it’s also creative, so the perfect blend for me.

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Ablation Techniques in Interventional Oncology

Author and Curator: Dror Nir, PhD

“Ablation is removal of material from the surface of an object by vaporization, chipping, or other erosive processes.”; WikipediA.

The use of ablative techniques in medicine is known for decades. By the late 90s, the ability to manipulate ablation sources and control their application to area of interest improved to a level that triggered their adaptation to cancer treatment. To date, ablation  is still a controversial treatment, yet steadily growing in it’s offerings to very specific cancer patients’ population.

The attractiveness in ablation as a form of cancer treatment is in the promise of minimal invasiveness, focused tissue destruction and better quality of life due to the ability to partially maintain viability of affected organs.  The main challenges preventing wider adaptation of ablative treatments are: the inability to noninvasively assess the level of cancerous tissue destruction during treatment; resulting in metastatic recurrence of the disease and the insufficient isolation of the treatment area from its surrounding.   This frequently results In addition, post-ablation salvage treatments are much more complicated. Since failed ablative treatment represents a lost opportunity to apply effective treatment to the primary tumor the current trend is to apply such treatments to low-grade cancers.

Nevertheless, the attractiveness of treating cancer in a focused way that preserves the long-term quality of life continuously feeds the development efforts and investments related to introduction of new and improved ablative treatments giving the hope that sometime in the future focused ablative treatment will reach its full potential.

The following paper reviews the main ablation techniques that are available for use today: Percutaneous image-guided ablation of bone and soft tissue tumours: a review of available techniques and protective measures.

Abstract

Background

Primary or metastatic osseous and soft tissue lesions can be treated by ablation techniques.

Methods

These techniques are classified into chemical ablation (including ethanol or acetic acid injection) and thermal ablation (including laser, radiofrequency, microwave, cryoablation, radiofrequency ionisation and MR-guided HIFU). Ablation can be performed either alone or in combination with surgical or other percutaneous techniques.

Results

In most cases, ablation provides curative treatment for benign lesions and malignant lesions up to 3 cm. Furthermore, it can be a palliative treatment providing pain reduction and local control of the disease, diminishing the tumor burden and mass effect on organs. Ablation may result in bone weakening; therefore, whenever stabilization is undermined, bone augmentation should follow ablation depending on the lesion size and location.

Conclusion

Thermal ablation of bone and soft tissues demonstrates high success and relatively low complication rates. However, the most common complication is the iatrogenic thermal damage of surrounding sensitive structures. Nervous structures are very sensitive to extremely high and low temperatures with resultant transient or permanent neurological damage. Thermal damage can cause normal bone osteonecrosis in the lesion’s periphery, surrounding muscular atrophy and scarring, and skin burns. Successful thermal ablation requires a sufficient ablation volume and thermal protection of the surrounding vulnerable structures.

Teaching points

Percutaneous ablations constitute a safe and efficacious therapy for treatment of osteoid osteoma.

Ablation techniques can treat painful malignant MSK lesions and provide local tumor control.

Thermal ablation of bone and soft tissues demonstrates high success and low complication rates.

Nerves, cartilage and skin are sensitive to extremely high and low temperatures.

Successful thermal ablation occasionally requires thermal protection of the surrounding structures.

For the purpose of this chapter we picked up three techniques:

Radiofrequency ablation

Straight or expandable percutaneously placed electrodes deliver a high-frequency alternating current, which causes ionic agitation with resultant frictional heat (temperatures of 60–100 ˚C) that produces protein denaturation and coagulation necrosis [8]. Concerning active protective techniques, all kinds of gas dissection can be performed. Hydrodissection is performed with dextrose 5 % (acts as an insulator as opposed to normal saline, which acts as a conductor). All kinds of skin cooling, thermal and neural monitoring can be performed.

 

Microwave ablation

Straight percutaneously placed antennae deliver electromagnetic microwaves (915 or 2,450 MHz) with resultant frictional heat (temperatures of 60–100 ˚C) that produces protein denaturation and coagulation necrosis [8]. Concerning active protective techniques, all kinds of gas dissection can be performed, whilst hydrodissection is usually avoided (MWA is based on agitation of water molecules for energy transmission). All kinds of skin cooling, thermal and neural monitoring can be performed.

Percutaneous ablation of malignant metastatic lesions is performed under imaging guidance, extended local sterility measures and antibiotic prophylaxis. Whenever the ablation zone is expected to extend up to 1 cm close to critical structures (e.g. the nerve root, skin, etc.), all the necessary thermal protection techniques should be applied (Fig. 3).

13244_2014_332_Fig3_HTML

a Painful soft tissue mass infiltrating the left T10 posterior rib. b A microwave antenna is percutaneously inserted inside the mass. Due to the proximity to the skin a sterile glove filled with cold water is placed over the skin. c CT axial scan 3 months

Irreversible Electroporation (IRE)

Each cell membrane point has a local transmembrane voltage that determines a dynamic phenomenon called electroporation (reversible or irreversible) [16]. Electroporation is manifested by specific transmembrane voltage thresholds related to a given pulse duration and shape. Thus, a threshold for an electronic field magnitude is defined and only cells with higher electric field magnitudes than this threshold are electroporated. IRE produces persistent nano-sized membrane pores compromising the viability of cells [16]. On the other hand, collagen and other supporting structures remain unaffected. The IRE generator produces direct current (25–45 A) electric pulses of high voltage (1,500–3,000 V).

Lastly we wish to highlight a method that is mostly used on patients diagnosed at intermediate or advanced clinical stages of Hepatocellular Carcinoma (HCC); transarterial chemoembolization  (TACE)

“Transcatheter arterial chemoembolization (also called transarterial chemoembolization or TACE) is a minimally invasive procedure performed in interventional radiology  to restrict a tumor’s blood supply. Small embolic particles coated with chemotherapeutic agents are injected selectively into an artery directly supplying a tumor. TACE derives its beneficial effect by two primary mechanisms. Most tumors within the liver are supplied by the proper hepatic artery, so arterial embolization preferentially interrupts the tumor’s blood supply and stalls growth until neovascularization. Secondly, focused administration of chemotherapy allows for delivery of a higher dose to the tissue while simultaneously reducing systemic exposure, which is typically the dose limiting factor. This effect is potentiated by the fact that the chemotherapeutic drug is not washed out from the tumor vascular bed by blood flow after embolization. Effectively, this results in a higher concentration of drug to be in contact with the tumor for a longer period of time. Park et al. conceptualized carcinogenesis of HCC as a multistep process involving parenchymal arterialization, sinusoidal capillarization, and development of unpaired arteries (a vital component of tumor angiogenesis). All these events lead to a gradual shift in tumor blood supply from portal to arterial circulation. This concept has been validated using dynamic imaging modalities by various investigators. Sigurdson et al. demonstrated that when an agent was infused via the hepatic artery, intratumoral concentrations were ten times greater compared to when agents were administered through the portal vein. Hence, arterial treatment targets the tumor while normal liver is relatively spared. Embolization induces ischemic necrosis of tumor causing a failure of the transmembrane pump, resulting in a greater absorption of agents by the tumor cells. Tissue concentration of agents within the tumor is greater than 40 times that of the surrounding normal liver.”; WikipediA

A recent open access research paper: Conventional transarterial chemoembolization versus drug-eluting bead transarterial chemoembolization for the treatment of hepatocellular carcinoma is discussing recent clinical approaches  related to this techniques.

Abstract

Background

To compare the overall survival of patients with hepatocellular carcinoma (HCC) who were treated with lipiodol-based conventional transarterial chemoembolization (cTACE) with that of patients treated with drug-eluting bead transarterial chemoembolization (DEB-TACE).

Methods

By an electronic search of our radiology information system, we identified 674 patients that received TACE between November 2002 and July 2013. A total of 520 patients received cTACE, and 154 received DEB-TACE. In total, 424 patients were excluded for the following reasons: tumor type other than HCC (n = 91), liver transplantation after TACE (n = 119), lack of histological grading (n = 58), incomplete laboratory values (n = 15), other reasons (e.g., previous systemic chemotherapy) (n = 114), or were lost to follow-up (n = 27). Therefore, 250 patients were finally included for comparative analysis (n = 174 cTACE; n = 76 DEB-TACE).

Results

There were no significant differences between the two groups regarding sex, overall status (Barcelona Clinic Liver Cancer classification), liver function (Child-Pugh), portal invasion, tumor load, or tumor grading (all p > 0.05). The mean number of treatment sessions was 4 ± 3.1 in the cTACE group versus 2.9 ± 1.8 in the DEB-TACE group (p = 0.01). Median survival was 409 days (95 % CI: 321–488 days) in the cTACE group, compared with 369 days (95 % CI: 310–589 days) in the DEB-TACE group (p = 0.76). In the subgroup of Child A patients, the survival was 602 days (484–792 days) for cTACE versus 627 days (364–788 days) for DEB-TACE (p = 0.39). In Child B/C patients, the survival was considerably lower: 223 days (165–315 days) for cTACE versus 226 days (114–335 days) for DEB-TACE (p = 0.53).

Conclusion

The present study showed no significant difference in overall survival between cTACE and DEB-TACE in patients with HCC. However, the significantly lower number of treatments needed in the DEB-TACE group makes it a more appealing treatment option than cTACE for appropriately selected patients with unresectable HCC.

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Imaging Technology in Cancer Surgery

Author and curator: Dror Nir, PhD

The advent of medical-imaging technologies such as image-fusion, functional-imaging and noninvasive tissue characterisation is playing an imperative role in answering this demand thus transforming the concept of personalized medicine in cancer into practice. The leading modality in that respect is medical imaging. To date, the main imaging systems that can provide reasonable level of cancer detection and localization are: CT, mammography, Multi-Sequence MRI, PET/CT and ultrasound. All of these require skilled operators and experienced imaging interpreters in order to deliver what is required at a reasonable level. It is generally agreed by radiologists and oncologists that in order to provide a comprehensive work-flow that complies with the principles of personalized medicine, future cancer patients’ management will heavily rely on computerized image interpretation applications that will extract from images in a standardized manner measurable imaging biomarkers leading to better clinical assessment of cancer patients.

As consequence of the human genome project and technological advances in gene-sequencing, the understanding of cancer advanced considerably. This led to increase in the offering of treatment options. Yet, surgical resection is still the leading form of therapy offered to patients with organ confined tumors. Obtaining “cancer free” surgical margins is crucial to the surgery outcome in terms of overall survival and patients’ quality of life/morbidity. Currently, a significant portion of surgeries ends up with positive surgical margins leading to poor clinical outcome and increase of costs. To improve on this, large variety of intraoperative imaging-devices aimed at resection-guidance have been introduced and adapted in the last decade and it is expected that this trend will continue.

The Status of Contemporary Image-Guided Modalities in Oncologic Surgery is a review paper presenting a variety of cancer imaging techniques that have been adapted or developed for intra-operative surgical guidance. It also covers novel, cancer-specific contrast agents that are in early stage development and demonstrate significant promise to improve real-time detection of sub-clinical cancer in operative setting.

Another good (free access) review paper is: uPAR-targeted multimodal tracer for pre- and intraoperative imaging in cancer surgery

Abstract

Pre- and intraoperative diagnostic techniques facilitating tumor staging are of paramount importance in colorectal cancer surgery. The urokinase receptor (uPAR) plays an important role in the development of cancer, tumor invasion, angiogenesis, and metastasis and over-expression is found in the majority of carcinomas. This study aims to develop the first clinically relevant anti-uPAR antibody-based imaging agent that combines nuclear (111In) and real-time near-infrared (NIR) fluorescent imaging (ZW800-1). Conjugation and binding capacities were investigated and validated in vitro using spectrophotometry and cell-based assays. In vivo, three human colorectal xenograft models were used including an orthotopic peritoneal carcinomatosis model to image small tumors. Nuclear and NIR fluorescent signals showed clear tumor delineation between 24h and 72h post-injection, with highest tumor-to-background ratios of 5.0 ± 1.3 at 72h using fluorescence and 4.2 ± 0.1 at 24h with radioactivity. 1-2 mm sized tumors could be clearly recognized by their fluorescent rim. This study showed the feasibility of an uPAR-recognizing multimodal agent to visualize tumors during image-guided resections using NIR fluorescence, whereas its nuclear component assisted in the pre-operative non-invasive recognition of tumors using SPECT imaging. This strategy can assist in surgical planning and subsequent precision surgery to reduce the number of incomplete resections.

INTRODUCTION
Diagnosis, staging, and surgical planning of colorectal cancer patients increasingly rely on imaging techniques that provide information about tumor biology and anatomical structures [1-3]. Single-photon emission computed tomography (SPECT) and positron emission tomography (PET) are preoperative nuclear imaging modalities used to provide insights into tumor location, tumor biology, and the surrounding micro-environment [4]. Both techniques depend on the recognition of tumor cells using radioactive ligands. Various monoclonal antibodies, initially developed as therapeutic agents (e.g. cetuximab, bevacizumab, labetuzumab), are labeled with radioactive tracers and evaluated for pre-operative imaging purposes [5-9]. Despite these techniques, during surgery the surgeons still rely mostly on their eyes and hands to distinguish healthy from malignant tissues, resulting in incomplete resections or unnecessary tissue removal in up to 27% of rectal cancer patients [10, 11]. Incomplete resections (R1) are shown to be a strong predictor of development of distant metastasis, local recurrence, and decreased survival of colorectal cancer patients [11, 12]. Fluorescence-guided surgery (FGS) is an intraoperative imaging technique already introduced and validated in the clinic for sentinel lymph node (SLN) mapping and biliary imaging [13]. Tumor-specific FGS can be regarded as an extension of SPECT/PET, using fluorophores instead of radioactive labels conjugated to tumor-specific ligands, but with higher spatial resolution than SPECT/PET imaging and real-time anatomical feedback [14]. A powerful synergy can be achieved when nuclear and fluorescent imaging modalities are combined, extending the nuclear diagnostic images with real-time intraoperative imaging. This combination can lead to improved diagnosis and management by integrating pre-intra and postoperative imaging. Nuclear imaging enables pre-operative evaluation of tumor spread while during surgery deeper lying spots can be localized using the gamma probe counter. The (NIR) fluorescent signal aids the surgeon in providing real-time anatomical feedback to accurately recognize and resect malignant tissues. Postoperative, malignant cells can be recognized using NIR fluorescent microscopy. Clinically, the advantages of multimodal agents in image-guided surgery have been shown in patients with melanoma and prostate cancer, but those studies used a-specific agents, following the natural lymph drainage pattern of colloidal tracers after peritumoral injection [15, 16]. The urokinase-type plasminogen activator receptor (uPAR) is implicated in many aspects of tumor growth and (micro) metastasis [17, 18]. The levels of uPAR are undetectable in normal tissues except for occasional macrophages and granulocytes in the uterus, thymus, kidneys and spleen [19]. Enhanced tumor levels of uPAR and its circulating form (suPAR) are independent prognostic markers for overall survival in colorectal cancer patients [20, 21]. The relatively selective and high overexpression of uPAR in a wide range of human cancers including colorectal, breast, and pancreas nominate uPAR as a widely applicable and potent molecular target [17,22]. The current study aims to develop a clinically relevant uPAR-specific multimodal agent that can be used to visualize tumors pre- and intraoperatively after a single injection. We combined the 111Indium isotope with NIR fluorophore ZW800-1 using a hybrid linker to an uPAR specific monoclonal antibody (ATN-658) and evaluated its performance using a pre-clinical SPECT system (U-SPECT-II) and a clinically-applied NIR fluorescence camera system (FLARE™).

Fig1 Fig2 Fig3

Robotic surgery is a growing trend as a form of surgery, specifically in urology. The following review paper propose a good discussion on the added value of imaging in urologic robotic surgery:

The current and future use of imaging in urological robotic surgery: a survey of the European Association of Robotic Urological Surgeons

 Abstract

Background

With the development of novel augmented reality operating platforms the way surgeons utilize imaging as a real-time adjunct to surgical technique is changing.

Methods

A questionnaire was distributed via the European Robotic Urological Society mailing list. The questionnaire had three themes: surgeon demographics, current use of imaging and potential uses of an augmented reality operating environment in robotic urological surgery.

Results

117 of the 239 respondents (48.9%) were independently practicing robotic surgeons. 74% of surgeons reported having imaging available in theater for prostatectomy 97% for robotic partial nephrectomy and 95% cystectomy. 87% felt there was a role for augmented reality as a navigation tool in robotic surgery.

Conclusions

This survey has revealed the contemporary robotic surgeon to be comfortable in the use of imaging for intraoperative planning it also suggests that there is a desire for augmented reality platforms within the urological community. Copyright © 2014 John Wiley & Sons, Ltd.

 Introduction

Since Röntgen first utilized X-rays to image the carpal bones of the human hand in 1895, medical imaging has evolved and is now able to provide a detailed representation of a patient’s intracorporeal anatomy, with recent advances now allowing for 3-dimensional (3D) reconstructions. The visualization of anatomy in 3D has been shown to improve the ability to localize structures when compared with 2D with no change in the amount of cognitive loading [1]. This has allowed imaging to move from a largely diagnostic tool to one that can be used for both diagnosis and operative planning.

One potential interface to display 3D images, to maximize its potential as a tool for surgical guidance, is to overlay them onto the endoscopic operative scene (augmented reality). This addresses, in part, a criticism often leveled at robotic surgery, the loss of haptic feedback. Augmented reality has the potential to mitigate this sensory loss by enhancing the surgeons visual cues with information regarding subsurface anatomical relationships [2].

Augmented reality surgery is in its infancy for intra-abdominal procedures due in large part to the difficulties of applying static preoperative imaging to a constantly deforming intraoperative scene [3]. There are case reports and ex vivo studies in the literature examining the technology in minimal access prostatectomy [3-6] and partial nephrectomy [7-10], but there remains a lack of evidence determining whether surgeons feel there is a role for the technology and if so for what procedures they feel it would be efficacious.

This questionnaire-based study was designed to assess first, the pre- and intra-operative imaging modalities utilized by robotic urologists; second, the current use of imaging intraoperatively for surgical planning; and finally whether there is a desire for augmented reality among the robotic urological community.

Methods

Recruitment

A web based survey instrument was designed and sent out, as part of a larger survey, to members of the EAU robotic urology section (ERUS). Only independently practicing robotic surgeons performing robot-assisted laparoscopic prostatectomy (RALP), robot-assisted partial nephrectomy (RAPN) and/or robotic cystectomy were included in the analysis, those surgeons exclusively performing other procedures were excluded. Respondents were offered no incentives to reply. All data collected was anonymous.

Survey design and administration

The questionnaire was created using the LimeSurvey platform (www.limesurvey.com) and hosted on their website. All responses (both complete and incomplete) were included in the analysis. The questionnaire was dynamic with the questions displayed tailored to the respondents’ previous answers.

When computing fractions or percentages the denominator was the number of respondents to answer the question, this number is variable due to the dynamic nature of the questionnaire.

Demographics

All respondents to the survey were asked in what country they practiced and what robotic urological procedures they performed. In addition to what procedures they performed surgeons were asked to specify the number of cases they had undertaken for each procedure.

 Current imaging practice

Procedure-specific questions in this group were displayed according to the operations the respondent performed. A summary of the questions can be seen in Appendix 1. Procedure-nonspecific questions were also asked. Participants were asked whether they routinely used the Tile Pro™ function of the da Vinci console (Intuitive Surgical, Sunnyvale, USA) and whether they routinely viewed imaging intra-operatively.

 Augmented reality

Before answering questions in this section, participants were invited to watch a video demonstrating an augmented reality platform during RAPN, performed by our group at Imperial College London. A still from this video can be seen in Figure 1. They were then asked whether they felt augmented reality would be of use as a navigation or training tool in robotic surgery.

f1

Figure 1. A still taken from a video of augmented reality robot assisted partial nephrectomy performed. Here the tumour has been painted into the operative view allowing the surgeon to appreciate the relationship of the tumour with the surface of the kidney

Once again, in this section, procedure-specific questions were displayed according to the operations the respondent performed. Only those respondents who felt augmented reality would be of use as a navigation tool were asked procedure-specific questions. Questions were asked to establish where in these procedures they felt an augmented reality environment would be of use.

Results

Demographics

Of the 239 respondents completing the survey 117 were independently practising robotic surgeons and were therefore eligible for analysis. The majority of the surgeons had both trained (210/239, 87.9%) and worked in Europe (215/239, 90%). The median number of cases undertaken by those surgeons reporting their case volume was: 120 (6–2000), 9 (1–120) and 30 (1–270), for RALP, robot assisted cystectomy and RAPN, respectively.

 

Contemporary use of imaging in robotic surgery

When enquiring about the use of imaging for surgical planning, the majority of surgeons (57%, 65/115) routinely viewed pre-operative imaging intra-operatively with only 9% (13/137) routinely capitalizing on the TilePro™ function in the console to display these images. When assessing the use of TilePro™ among surgeons who performed RAPN 13.8% (9/65) reported using the technology routinely.

When assessing the imaging modalities that are available to a surgeon in theater the majority of surgeons performing RALP (74%, 78/106)) reported using MRI with an additional 37% (39/106) reporting the use of CT for pre-operative staging and/or planning. For surgeons performing RAPN and robot-assisted cystectomy there was more of a consensus with 97% (68/70) and 95% (54/57) of surgeons, respectively, using CT for routine preoperative imaging (Table 1).

Table 1. Which preoperative imaging modalities do you use for diagnosis and surgical planning?

  CT MRI USS None Other
RALP (n = 106) 39.8% 73.5% 2% 15.1% 8.4%
(39) (78) (3) (16) (9)
RAPN (n = 70) 97.1% 42.9% 17.1% 0% 2.9%
(68) (30) (12) (0) (2)
Cystectomy (n = 57) 94.7% 26.3% 1.8% 1.8% 5.3%
(54) (15) (1) (1) (3)

Those surgeons performing RAPN were found to have the most diversity in the way they viewed pre-operative images in theater, routinely viewing images in sagittal, coronal and axial slices (Table 2). The majority of these surgeons also viewed the images as 3D reconstructions (54%, 38/70).

Table 2. How do you typically view preoperative imaging in the OR? 3D recons = three-dimensional reconstructions

  Axial slices (n) Coronal slices (n) Sagittal slices (n) 3D recons. (n) Do not view (n)  
RALP (n = 106) 49.1% 44.3% 31.1% 9.4% 31.1%
(52) (47) (33) (10) (33)
RAPN (n = 70) 68.6% 74.3% 60% (42) 54.3% 0%
(48) (52) (38) (0)
Cystectomy (n = 57) 70.2% 52.6% 50.9% 21.1% 8.8%
(40) (30) (29) (12) (5)

The majority of surgeons used ultrasound intra-operatively in RAPN (51%, 35/69) with a further 25% (17/69) reporting they would use it if they had access to a ‘drop-in’ ultrasound probe (Figure 2).

f2

Figure 2. Chart demonstrating responses to the question – Do you use intraoperative ultrasound for robotic partial nephrectomy?

Desire for augmented reality

Overall, 87% of respondents envisaged a role for augmented reality as a navigation tool in robotic surgery and 82% (88/107) felt that there was an additional role for the technology as a training tool.

The greatest desire for augmented reality was among those surgeons performing RAPN with 86% (54/63) feeling the technology would be of use. The largest group of surgeons felt it would be useful in identifying tumour location, with significant numbers also feeling it would be efficacious in tumor resection (Figure 3).

f3

Figure 3. Chart demonstrating responses to the question – In robotic partial nephrectomy which parts of the operation do you feel augmented reality image overlay would be of assistance?

When enquiring about the potential for augmented reality in RALP, 79% (20/96) of respondents felt it would be of use during the procedure, with the largest group feeling it would be helpful for nerve sparing 65% (62/96) (Figure 4). The picture in cystectomy was similar with 74% (37/50) of surgeons believing augmented reality would be of use, with both nerve sparing and apical dissection highlighted as specific examples (40%, 20/50) (Figure 5). The majority also felt that it would be useful for lymph node dissection in both RALP and robot assisted cystectomy (55% (52/95) and 64% (32/50), respectively).

f4

Figure 4. Chart demonstrating responses to the question – In robotic prostatectomy which parts of the operation do you feel augmented reality image overlay would be of assistance?

f5

Figure 5. Chart demonstrating responses to the question – In robotic cystectomy which parts of the operation do you feel augmented reality overlay technology would be of assistance?

Discussion

The results from this study suggest that the contemporary robotic surgeon views imaging as an important adjunct to operative practice. The way these images are being viewed is changing; although the majority of surgeons continue to view images as two-dimensional (2D) slices a significant minority have started to capitalize on 3D reconstructions to give them an improved appreciation of the patient’s anatomy.

This study has highlighted surgeons’ willingness to take the next step in the utilization of imaging in operative planning, augmented reality, with 87% feeling it has a role to play in robotic surgery. Although there appears to be a considerable desire for augmented reality, the technology itself is still in its infancy with the limited evidence demonstrating clinical application reporting only qualitative results [3, 7, 11, 12].

There are a number of significant issues that need to be overcome before augmented reality can be adopted in routine clinical practice. The first of these is registration (the process by which two images are positioned in the same coordinate system such that the locations of corresponding points align [13]). This process has been performed both manually and using automated algorithms with varying degrees of accuracy [2, 14]. The second issue pertains to the use of static pre-operative imaging in a dynamic operative environment; in order for the pre-operative imaging to be accurately registered it must be deformable. This problem remains as yet unresolved.

Live intra-operative imaging circumvents the problems of tissue deformation and in RAPN 51% of surgeons reported already using intra-operative ultrasound to aid in tumour resection. Cheung and colleagues [9] have published an ex vivo study highlighting the potential for intra-operative ultrasound in augmented reality partial nephrectomy. They report the overlaying of ultrasound onto the operative scene to improve the surgeon’s appreciation of the subsurface tumour anatomy, this improvement in anatomical appreciation resulted in improved resection quality over conventional ultrasound guided resection [9]. Building on this work the first in vivo use of overlaid ultrasound in RAPN has recently been reported [10]. Although good subjective feedback was received from the operating surgeon, the study was limited to a single case demonstrating feasibility and as such was not able to show an outcome benefit to the technology [10].

RAPN also appears to be the area in which augmented reality would be most readily adopted with 86% of surgeons claiming they see a use for the technology during the procedure. Within this operation there are two obvious steps to augmentation, anatomical identification (in particular vessel identification to facilitate both routine ‘full clamping’ and for the identification of secondary and tertiary vessels for ‘selective clamping’ [15]) and tumour resection. These two phases have different requirements from an augmented reality platform; the first phase of identification requires a gross overview of the anatomy without the need for high levels of registration accuracy. Tumor resection, however, necessitates almost sub-millimeter accuracy in registration and needs the system to account for the dynamic intra-operative environment. The step of anatomical identification is amenable to the use of non-deformable 3D reconstructions of pre-operative imaging while that of image-guided tumor resection is perhaps better suited to augmentation with live imaging such as ultrasound [2, 9, 16].

For RALP and robot-assisted cystectomy the steps in which surgeons felt augmented reality would be of assistance were those of neurovascular bundle preservation and apical dissection. The relative, perceived, efficacy of augmented reality in these steps correlate with previous examinations of augmented reality in RALP [17, 18]. Although surgeon preference for utilizing augmented reality while undertaking robotic prostatectomy has been demonstrated, Thompson et al. failed to demonstrate an improvement in oncological outcomes in those patients undergoing AR RALP [18].

Both nerve sparing and apical dissection require a high level of registration accuracy and a necessity for either live imaging or the deformation of pre-operative imaging to match the operative scene; achieving this level of registration accuracy is made more difficult by the mobilization of the prostate gland during the operation [17]. These problems are equally applicable to robot-assisted cystectomy. Although guidance systems have been proposed in the literature for RALP [3-5, 12, 17], none have achieved the level of accuracy required to provide assistance during nerve sparing. In addition, there are still imaging challenges that need to be overcome. Although multiparametric MRI has been shown to improve decision making in opting for a nerve sparing approach to RALP [19] the imaging is not yet able to reliably discern the exact location of the neurovascular bundle. This said, significant advances are being made with novel imaging modalities on the horizon that may allow for imaging of the neurovascular bundle in the near future [20].

 

Limitations

The number of operations included represents a significant limitation of the study, had different index procedures been chosen different results may have been seen. This being said the index procedures selected were chosen as they represent the vast majority of uro-oncological robotic surgical practice, largely mitigating for this shortfall.

Although the available ex vivo evidence suggests that introducing augmented reality operating environments into surgical practice would help to improve outcomes [9, 21] the in vivo experience to date is limited to small volume case series reporting feasibility [2, 3, 14]. To date no study has demonstrated an in vivo outcome advantage to augmented reality guidance. In addition to this limitation augmented reality has been demonstrated to increased rates of inattention blindness among surgeons suggesting there is a trade-off between increasing visual information and the surgeon’s ability to appreciate unexpected operative events [21].

 

Conclusions

This survey shows the contemporary robotic surgeon to be comfortable with the use of imaging to aid intra-operative planning; furthermore it highlights a significant interest among the urological community in augmented reality operating platforms.

Short- to medium-term development of augmented reality systems in robotic urology surgery would be best performed using RAPN as the index procedure. Not only was this the operation where surgeons saw the greatest potential benefits, but it may also be the operation where it is most easily achievable by capitalizing on the respective benefits of technologies the surgeons are already using; pre-operative CT for anatomical identification and intra-operative ultrasound for tumour resection.

 

Conflict of interest

None of the authors have any conflicts of interest to declare.

Appendix 1

Question Asked Question Type
Demographics
In which country do you usually practise? Single best answer
Which robotic procedures do you perform?* Single best answer
Current Imaging Practice
What preoperative imaging modalities do you use for the staging and surgical planning in renal cancer? Multiple choice
How do you typically view preoperative imaging in theatre for renal cancer surgery? Multiple choice
Do you use intraoperative ultrasound for partial nephrectomy? Yes or No
What preoperative imaging modalities do you use for the staging and surgical planning in prostate cancer? Multiple choice
How do you typically view preoperative imaging in theatre for prostate cancer? Multiple choice
Do you use intraoperative ultrasound for robotic partial nephrectomy? Yes or No
Which preoperative imaging modality do you use for staging and surgical planning in muscle invasive TCC? Multiple choice
How do you typically view preoperative imaging in theatre for muscle invasive TCC? Multiple choice
Do you routinely refer to preoperative imaging intraoperativley? Yes or No
Do you routinely use Tilepro intraoperativley? Yes or No
Augmented Reality
Do you feel there is a role for augmented reality as a navigation tool in robotic surgery? Yes or No
Do you feel there is a role for augmented reality as a training tool in robotic surgery? Yes or No
In robotic partial nephrectomy which parts of the operation do you feel augmented reality image overlay technology would be of assistance? Multiple choice
In robotic nephrectomy which parts of the operation do you feel augmented reality image overlay technology would be of assistance? Multiple choice
In robotic prostatectomy which parts of the operation do you feel augmented reality image overlay technology would be of assistance? Multiple choice
Would augmented reality guidance be of use in lymph node dissection in robotic prostatectomy? Yes or No
In robotic cystectomy which parts of the operation do you feel augmented reality image overlay technology would be of assistance? Multiple choice
Would augmented reality guidance be of use in lymph node dissection in robotic cystectomy? Yes or No
*The relevant procedure related questions were displayed based on the answer to this question

References

1. Foo J-L, Martinez-Escobar M, Juhnke B, et al.Evaluating mental workload of two-dimensional and three-dimensional visualization for anatomical structure localization. J Laparoendosc Adv Surg Tech A 2013; 23(1):65–70.

2. Hughes-Hallett A, Mayer EK, Marcus HJ, et al.Augmented reality partial nephrectomy: examining the current status and future perspectives. Urology 2014; 83(2): 266–273.

3. Sridhar AN, Hughes-Hallett A, Mayer EK, et al.Image-guided robotic interventions for prostate cancer. Nat Rev Urol 2013; 10(8): 452–462.

4. Cohen D, Mayer E, Chen D, et al.Eddie’ Augmented reality image guidance in minimally invasive prostatectomy. Lect Notes Comput Sci 2010; 6367: 101–110.

5. Simpfendorfer T, Baumhauer M, Muller M, et al.Augmented reality visualization during laparoscopic radical prostatectomy. J Endourol 2011; 25(12): 1841–1845.

6. Teber D, Simpfendorfer T, Guven S, et al.In vitro evaluation of a soft-tissue navigation system for laparoscopic prostatectomy. J Endourol 2010; 24(9): 1487–1491.

7. Teber D, Guven S, Simpfendörfer T, et al.Augmented reality: a new tool to improve surgical accuracy during laparoscopic partial nephrectomy? Preliminary in vitro and in vivo Eur Urol 2009; 56(2): 332–338.

8. Pratt P, Mayer E, Vale J, et al.An effective visualisation and registration system for image-guided robotic partial nephrectomy. J Robot Surg 2012; 6(1): 23–31.

9. Cheung CL, Wedlake C, Moore J, et al.Fused video and ultrasound images for minimally invasive partial nephrectomy: a phantom study. Med Image Comput Comput Assist Interv 2010; 13(Pt 3): 408–415.

10. Hughes-Hallett A, Pratt P, Mayer E, et al.Intraoperative ultrasound overlay in robot-assisted partial nephrectomy: first clinical experience. Eur Urol 2014; 65(3): 671–672.

11. Nakamura K, Naya Y, Zenbutsu S, et al.Surgical navigation using three-dimensional computed tomography images fused intraoperatively with live video. J Endourol 2010; 24(4): 521–524.

12. Ukimura O, Gill IS. Imaging-assisted endoscopic surgery: Cleveland clinic experience. J Endourol2008; 22(4):803–809.

13. Altamar HO, Ong RE, Glisson CL, et al.Kidney deformation and intraprocedural registration: a study of elements of image-guided kidney surgery. J Endourol 2011; 25(3): 511–517.

14. Nicolau S, Soler L, Mutter D, Marescaux J. Augmented reality in laparoscopic surgical oncology. Surg Oncol2011; 20(3): 189–201.

15. Ukimura O, Nakamoto M, Gill IS. Three-dimensional reconstruction of renovascular-tumor anatomy to facilitate zero-ischemia partial nephrectomy. Eur Urol2012; 61(1): 211–217.

16. Pratt P, Hughes-Hallett A, Di Marco A, et al. Multimodal reconstruction for image-guided interventions. In:Yang GZ, Darzi A (eds) Proceedings of the Hamlyn symposium on medical robotics: London. 2013; 59–61.

17. Mayer EK, Cohen D, Chen D, et al.Augmented reality image guidance in minimally invasive prostatectomy. Eur Urol Supp 2011; 10(2): 300.

18. Thompson S, Penney G, Billia M, et al.Design and evaluation of an image-guidance system for robot-assisted radical prostatectomy. BJU Int 2013; 111(7): 1081–1090.

19. Panebianco V, Salciccia S, Cattarino S, et al.Use of multiparametric MR with neurovascular bundle evaluation to optimize the oncological and functional management of patients considered for nerve-sparing radical prostatectomy. J Sex Med 2012; 9(8): 2157–2166.

20. Rai S, Srivastava A, Sooriakumaran P, Tewari A. Advances in imaging the neurovascular bundle. Curr Opin Urol2012; 22(2): 88–96.

21. Dixon BJ, Daly MJ, Chan H, et al.Surgeons blinded by enhanced navigation: the effect of augmented reality on attention. Surg Endosc 2013; 27(2): 454–461.

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Palliative Care

Writer and Curator: Larry H. Bernstein, MD., FCAP

http://www.caregiverslibrary.org/caregivers-resources/grp-end-of-life-issues/hsgrp-hospice/hospice-vs-palliative-care-article.aspx

The differences between hospice and palliative care.

Hospice care and palliative care are very similar when it comes to the most important issue for dying people: care. Most people have heard of hospice care and have a general idea of what services hospice provides. What they don’t know or what may become confusing is that hospice provides “palliative care,” and that palliative care is both a method of administering “comfort” care and increasingly, an administered system of palliative care offered most prevalently by hospitals. As an adjunct or supplement to some of the more “traditional” care options, both hospice and palliative care protocols call for patients to receive a combined approach where medications, day-to-day care, equipment, bereavement counseling, and symptom treatment are administered through a single program. Where palliative care programs and hospice care programs differ greatly is in the care location, timing, payment, and eligibility for services.
Hospice

Hospice programs far outnumber palliative care programs. Generally, once enrolled through a referral from the primary care physician, a patient’s hospice care program, which is overseen by a team of hospice professionals, is administered in the home. Hospice often relies upon the family caregiver, as well as a visiting hospice nurse. While hospice can provide round-the-clock care in a nursing home, a specially equipped hospice facility, or, on occasion, in a hospital, this is not the norm.

Palliative Care

Palliative care teams are made up of doctors, nurses, and other professional medical caregivers, often at the facility where a patient will first receive treatment. These individuals will administer or oversee most of the ongoing comfort-care patients receive. While palliative care can be administered in the home, it is most common to receive palliative care in an institution such as a hospital, extended care facility, or nursing home that is associated with a palliative care team.

Hospice

You must generally be considered to be terminal or within six months of death to be eligible for most hospice programs or to receive hospice benefits from your insurance.

Palliative Care

There are no time restrictions. Palliative care can be received by patients at any time, at any stage of illness whether it be terminal or not.

Hospice

Before considering hospice, it is important to check on policy limits for payment. While hospice can be considered an all-inclusive treatment in terms of payment (hospice programs cover almost all expenses) insurance coverage for hospice can vary. Some hospice programs offer subsidized care for the economically disadvantaged, or for patients not covered under their own insurance. Many hospice programs are covered under Medicare.

Palliative Care

Since this service will generally be administered through your hospital or regular medical provider, it is likely that it is covered by your regular medical insurance. It is important to note, however, that each item will be billed separately, just as they are with regular hospital and doctor visits. If you receive outpatient palliative care, prescriptions will be billed separately and are only covered as provided by your regular insurance. In-patient care however, often does cover prescription charges. For more details, check with your insurance company, doctor, or hospital administration.

Hospice

Most programs concentrate on comfort rather than aggressive disease abatement. By electing to forego extensive life-prolonging treatment, hospice patients can concentrate on getting the most out of the time they have left, without some of the negative side-effects that life prolonging treatments can have. Most hospice patients can achieve a level of comfort that allows them to concentrate on the emotional and practical issues of dying.

Palliative Care

Since there are no time limits on when you can receive palliative care, it acts to fill the gap for patients who want and need comfort at any stage of any disease, whether terminal or chronic. In a palliative care program, there is no expectation that life-prolonging therapies will be avoided.

It is important to note, however, that there will be exceptions to the general precepts outlined. There are some hospice programs that will provide life-prolonging treatments, and there are some palliative care programs that concentrate mostly on end-of-life care. Consult your physician or care-administrator for the best service for you.

Reprinted from “Hospice vs. Palliative Care,” by Ann Villet-Lagomarsino. Educational Broadcasting Corporation/Public Affairs Television, Inc. Reprinted with permission.

http://www.webmd.com/palliative-care/palliative-care-mr

For the last thirty years, palliative care has been provided by hospice programs for dying Americans. Currently these programs serve more than 1 million patients and their families each year.

Now this very same approach to care is being used by other health care providers, including teams in hospitals, nursing facilities and home health agencies in combination with other medical treatments to help people who are seriously ill.

To palliate means to make comfortable by treating a person’s symptoms from an illness. Hospice and palliative care both focus on helping a person be comfortable by addressing issues causing physical or emotional pain, or suffering. Hospice and other palliative care providers have teams of people working together to provide care. The goals of palliative care are to improve the quality of a seriously ill person’s life and to support that person and their family during and after treatment.

Hospice focuses on relieving symptoms and supporting patients with a life expectancy of months not years, and their families. However, palliative care may be given at any time during a patient’s illness, from diagnosis on. Most hospices have a set of defined services, team members and rules and regulations. Some hospices provide palliative care as a separate program or service, which can be very confusing to patients and families. The list of questions below provides answers to common questions about the difference between hospice and palliative care.

http://www.nlm.nih.gov/medlineplus/ency/patientinstructions/000536.htm

The goal of palliative care is to help patients with serious illnesses feel better. It prevents or treats symptoms and side effects of disease and treatment. Palliative care also treats emotional, social, practical, and spiritual problems that illnesses can bring up. When patients feel better in these areas, they have an improved quality of life.

Palliative care can be given at the same time as treatments meant to cure or treat the disease. You may get palliative care when the illness is diagnosed, throughout treatment, during follow-up, and at the end of life.

Who gives palliative care?

Any health care provider can give palliative care. But some providers specialize in it. Palliative care may be given by:

  • A team of doctors
  • Nurses
  • Registered dietitians
  • Social workers
  • Psychologists
  • Massage therapists
  • Chaplains

Palliative care may be offered by hospitals, home care agencies, cancer centers, and long term care facilities. Your doctor or hospital can give you the names of palliative care specialists near you.

A serious illness affects more than just the body. It touches all areas of a person’s life, as well as that person’s family members’ lives. Palliative care can address these effects of a person’s illness.

Physical problems. Symptoms or side effects include:

Treatments may include:

  • Medicine
  • Nutritional guidance
  • Physical therapy
  • Occupational therapy
  • Integrative therapies

Emotional, social, and coping problems. Patients and their families face stress during illness that can lead to fear, anxiety, hopelessness, or depression. Family members may take on care giving, even if they also have jobs and other duties.

Treatments may include:

  • Counseling
  • Support groups
  • Family meetings
  • Referrals to mental health providers

Practical problems. Some of the problems brought on by illness are practical, such as money- or job-related problems, insurance questions, and legal issues. A palliative care team may:

  • Explain complex medical forms or help families understand treatment choices
  • Provide or refer families to financial counseling
  • Help connect you to resources for transportation or housing

Spiritual issues. When people are challenged by illness, they may look for meaning or question their faith. A palliative care team may help patients and families explore their beliefs and values so they can move toward acceptance and peace.

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Cancer Biomarkers [11.3.2.3]

Writer and Curator: Larry H. Bernstein, MD, FCAP

Cancer Biomarkers

This discussion is extracted from the Special Section – Cancer Biomarker Update – in May Arch Pathology and Lab Med.  It is not intended to be complete, but it has quite timely content.  There are three articles I shall cover.

11.3.2.3 Cancer Biomarkers

11.3.2.3.1 Cancer Biomarkers in Structured Data Reporting

11.3.2.3.2 Cancer Biomarkers in Myeloid Malignancies

11.3.2.3.3 National Comprehensive Cancer Network Consensus on Use of Cancer Biomarkers

Cancer Biomarkers – The Role of Structured Data Reporting
Simpson,  RW; Berman, MA; Foulis PR, et al.
Arch Pathol Lab Med. 2015;139:587–593
http://dx.doi.org:/10.5858/arpa.2014-0082-RA

The College of American Pathologists has been producing cancer protocols since 1986 to aid pathologists in the diagnosis and reporting of cancer cases. Many pathologists use the included cancer case summaries as templates for dictation/data entry into the final pathology report. These summaries are now available in a computer-readable format with structured data elements for interoperability, packaged as ‘‘electronic cancer checklists.’’ Most major vendors of anatomic pathology reporting software support this model. Objectives.—To outline the development and advantages of structured electronic cancer reporting using the electronic cancer checklist model, and to describe its extension to cancer biomarkers and other aspects of cancer reporting. Data Sources.—Peer-reviewed literature and internal records of the College of American Pathologists. Conclusions.—Accurate and usable cancer biomarker data reporting will increasingly depend on initial capture of this information as structured data. This process will support the standardization of data elements and biomarker terminology, enabling the meaningful use of these datasets by pathologists, clinicians, tumor registries, and patients.

Narrative Versus Structured Data Reporting Clinical laboratory reports typically consist of discrete data elements with structured qualitative or quantitative information, often using standardized laboratory methods, data elements, and units. When discrete data elements are electronically transmitted to external clinical information systems, the transmitted information may be annotated with one or more terminologies such as Systematized Nomenclature of Medicine Clinical Terms (SNOMED CT)4 and Logical Observation Identifiers Names and Codes (LOINC),5 although the consistent application of such codes to structured laboratory data is not yet an interoperable standard. Because the structure of clinical laboratory data tends to be fixed and standardized before the point of data entry, reporting these data elements in a tabular synoptic format is a relatively simple process. The report output may not include all data collected (eg, methodologic details), but clinically relevant data can be easily extracted by computer algorithm and automatically reported in easily readable format (including custom text, result explanations, and test value trends).

Anatomic pathology reports, by contrast, have traditionally been narrative and recorded as unstructured or partially structured fields of text. Unfortunately, narrative reporting often lacks consistency in organization, content, units, terminology, and completeness.6–8 These structural inconsistencies create difficulties in finding and understanding clinically important data and increase the chance of omitting key data elements and misinterpreting information present in the narrative. This is particularly problematic when clinicians encounter reports from multiple pathology laboratories or when patients receive care at multiple institutions. Narrative reporting has equally negative effects on computer readability; the ability of computers to correctly parse and classify information contained in a narrative report is imperfect even when using advanced natural language processing software designed specifically for anatomic pathology.9,10 Natural language processing–parsed text must always undergo human review, editing, and signoff before release for patient care or research.

Ensuring consistency and readability of cancer and biomarker reports requires a reporting solution integrated into the pathologist workflow that supports entry of standardized data directly into a laboratory information system and/or electronic health record system. These systems can produce highly readable synoptic reports and can include computer-based report validation of standardized data elements to reduce or eliminate the chance of omitting required data elements. With the transition from narrative to synoptic reporting for cancer cases, many laboratories have been using modified CCPs or locally developed templates or macros, which may or may not contain all required data elements. This common mode of data entry fits well into the pathologist workflow and can result in organized, highly readable synoptic reports, but generally results in information stored as text in a single large data field. Even when results are entered as discrete data elements, subsequent storage mechanisms usually result in nonstructured text or nonstandard custom data fields in a local computer system. Unfortunately, narrative and nonstandardized data sets are very difficult to reliably aggregate and analyze for laboratory quality assurance, research, or cancer registry surveillance. Such aggregated data also remain relatively unreliable because of changes in information systems. Many of these issues can be eliminated by entering and reporting structured data with standardized electronic templates.

CAP eCC History, Development, and Adoption Efforts to bring structure to cancer pathology reporting began in the late 1980s and early 1990s1,11,12 with publication of templates that were the precursors of the current CCPs (Table). The primary goal was to improve the care of cancer patients by improving the reporting rate of clinically important data elements. The checklist approach was adopted to help standardize terminology and ensure all relevant data elements are reported. The 66 current CCPs, 3 new cancer biomarker templates,13–16 and 85 eCC templates represent the evolution of the original 1986 CCP model. The CCPs and templates are created and maintained through the ongoing work of the CAP Cancer and Cancer Biomarker Reporting Committees. The CCPs are widely adopted by laboratories and used for accreditation purposes by the American College of Surgeons–Commission on Cancer17 and CAP Laboratory Accreditation Program.18

During the past several years, the CAP has worked to create standardized pathology reporting templates that enable individual pathologists and software vendors to capture, store, retrieve, transmit, and analyze diagnostic cancer pathology information. Electronic versions of these templates, the eCCs, are based on consistent structured data representation, which enables simple yet robust computerization of cancer pathology data elements suitable for patient care, cancer registry transmission, and research. Synoptic reports are not the same as ‘‘structured data.’’ Although synoptic reports are formatted for optimum human readability and understanding, they consist of textbased questions and answers (ideally one pair per line) that present problems for computer readability and interoperability. Structured data, by contrast, refers to representation of data elements in a computer-readable data exchange format such as XML. The structured data model used by eCC XML templates assigns a unique identifier (a composite key) to every question and answer choice, template, section, and note listed in the template. Composite keys are used throughout the entire eCC life cycle to transmit the precise identity of each data element and its origin in a specific version of an eCC template.

The CAP eCC model has been implemented province wide by Cancer Care Ontario through their multiyear synoptic pathology reporting and change-management project.19–21 The Canadian Partnership Against Cancer is also currently working with several other Canadian provinces to implement population-level electronic synoptic reporting based on the CAP eCC. The Cancer Care Ontario project has shown that there is high acceptability among pathologists and clinicians22 and that data are usable for the secondary needs of tumor registries,20 but there remains room for improvement. For instance, both the Reporting Pathology Protocols project reports23–25 and the Cancer Care Ontario implementation reports22 suggest that pathologists require more time to complete the reporting task.

While this may meet quality and data reporting needs, it remains a potential barrier to acceptance. Automated human-readable report generation also could be improved, especially in terms of creating best practice guidelines for report output. Both data entry and report generation have traditionally been supported by laboratory information systems vendors, but the success of implementation has varied, and often significant effort is required to modify the resultant human readable report to satisfy local clinical needs. Because the final report remains most important for patient care, the CAP Diagnostic Intelligence in Health Information Technology and Pathology Electronic Reporting committees have initiated work on creating and promoting a standardized data structure within cancer pathology reports.

Abbreviated CAP eCC History and Milestones

2010–2012 CCO successfully implements population level electronic synoptic reporting in nearly all disease sites based on 2010 CAP eCC standards, which include AJCC 7th edition TNM staging; 97% of labs report using structured data from eCC.20

2010 CAP Laboratory Accreditation Program begins to survey institutions for inclusion of required CCP data elements in AP reports.38

2010 NAACCR Pathology Data Workgroup develops implementation guide to assist with CAP eCC-based transmissions of cancer data to central cancer registries.39

2011 CCO user acceptability data demonstrate high level of acceptance for eCC-derived synoptic reports among clinicians and pathologists.22

2012 CAP forms the multi-organizational Cancer Biomarker Reporting Workgroup, tasked to produce standardized reporting templates for breast, colorectal, and lung cancer biomarkers.40

2013 eCC-based reporting in Ontario is used to improve quality and practice performance.20

2013 The first cancer biomarker templates are produced for breast, colorectal, and lung cancer.13–16 They are available on the http://www.cap.org/cancerprotocols Web site in Word and PDF format (accessed April 28, 2014). The eCC versions are available through CAP.

2013 Launch of CAP eFRM, a software product to aid vendor integration of eCCs into AP-LIS systems or for use as a standalone product.

2014 By December 2013, CAP is maintaining current versions of 66 CCPs, 3 cancer biomarker templates, and 85 corresponding eCC templates.

References

13. Cagle PT, Sholl LM, Lindeman NI, et al. Template for reporting results of biomarker testing of specimens from patients with non–small cell carcinoma of the lung. Arch Pathol Lab Med. 2014; 138(2):171–174. http://dx.doi.org:/10.5858/arpa.2013-0232-CP

14. Cagle PT, Allen TC, Olsen RJ. Lung cancer biomarkers: present status and future developments. Arch Pathol Lab Med. 2013; 137(9):1191–1198.
http://dx.doi.org:/10.5858/arpa.2013-0319-CR

15. Bartley AN, Hamilton SR, Alsabeh R, et al. Template for reporting results of biomarker testing of specimens from patients with carcinoma of the colon and rectum. Arch Pathol Lab Med. 2014; 138(2):166–170.
http://dx.doi.org:/10.5858/arpa.2013-0231-CP

16. Fitzgibbons PL, Dillon DA, Alsabeh R, et al. Template for reporting results of biomarker testing of specimens from patients with carcinoma of the breast. Arch Pathol Lab Med. 2014; 138(5):595–601.
http://dx.doi.org:/10.5858/arpa.2013-0566-CP

20. Srigley J, Lankshear S, Brierley J, et al. Closing the quality loop: facilitating improvement in oncology practice through timely access to clinical performance indicators. J Oncol Pract. 2013; 9(5):e255–e261. http://dx.doi.org:/10.1200/JOP.2012.000818

22. Lankshear S, Srigley J, McGowan T, Yurcan M, Sawka C. Standardized synoptic cancer pathology reports—so what and who cares?: a population-based satisfaction survey of 970 pathologists, surgeons, and oncologists. Arch Pathol Lab Med. 2013; 137(11):1599–1602.
http://dx.doi.org:/10.5858/arpa.2012-0656-OA

38. College of American Pathologists. CAP cancer protocols frequently asked questions. http://www.cap.org/apps//cap.portal
39. Klein WT, Havener LA, eds. Standards for Cancer Registries Volume V: Pathology Laboratory Electronic Reporting, Version 4.0. Springfield, IL: North American Association of Central Cancer Registries; 2011:1–310.
40. Fitzgibbons PL, Lazar AJ, Spencer S. Introducing new College of American Pathologists reporting templates for cancer biomarkers. Arch Pathol Lab Med. 2014; 138(2):157–158. http://dx.doi.org:/10.5858/arpa.2013-0233-ED

41. Amin MB. The 2009 version of the cancer protocols of the College of American Pathologists. Arch Pathol Lab Med. 2010; 134(3):326–330.
http://dx.doi.org:/10. 1043/1543-2165-134.3.326.

Figure 1. (not shown) Narrative versus synoptic versus structured reporting of breast biomarker testing (excerpts). The narrative row shows a portion of a dictated biomarker report. The synoptic row satisfies the College of American Pathologists (CAP) synoptic reporting requirements, but is not computer readable.

 

 

Figure 2. (not shown) The College of American Pathologists (CAP) Cancer Protocols (CCPs) are developed by the CAP Cancer Committee. Each CCP is reformulated as question/answer structures, entered into the CAP electronic Cancer Checklist (eCC) Template Editor (not shown), and stored in the eCC template database. The eCC files in XML format are produced from this database and delivered to vendors of anatomic pathology/laboratory information system (LIS) software systems. Vendors convert the eCC files into data entry form implementations using their local technologies. In addition, most vendors create eCC-based templates for creating synoptic reports. When pathologists enter data into the eCC-based data-entry forms, the vendor software is able to run validation checks such as assessing whether all CCP-required data elements are recorded. Synoptic reports are developed from the eCC-derived data and delivered to health care providers for patient care. The eCC-structured data is stored in the vendor database, where it can be transmitted in interoperable format to other computer systems. Secondary uses of eCC-based data include cancer registry reporting, quality assurance, biospecimen annotation, research, decision support, and financial reporting. The horizontal arrows involve the exchange of eCC composite keys, preserving the fidelity of the data as part of an eCC template, providing the foundation for interoperable data transmission formats, and enabling the regeneration of eCC datasets in the exact format in which they were recorded. Activity columns that directly impact health care activities are shaded in light blue. Abbreviation: EHR, electronic health record.

Future The use of standardized, structured data elements is foundational for the development of improved reporting and clinical decision support for biomarker results. Clinicians are currently faced with synthesizing data from multiple narrative reports to decide on treatment options. Often these narrative reports are from different laboratories with very different report formats and include variable methodologic details, all of which hinders understanding of important results. For biomarkers that determine a patient’s eligibility for specific drugs, a computer-generated report that presents test results in a tabular form, similar to antibiotic susceptibility testing, may be desirable. This reporting method would allow for display of biomarker test results over time and could also link to other databases.

Structured data allows for clinical decision support such that the report displays only eligible drugs, or the report displays a note stating that a test result suggests a patient is not eligible for a specific drug. Using standardized terminology allows these rules to be the same between institutions, even if electronic health record system vendors use different means of implementation.

Figure 3. (not shown) College of American Pathologists electronic Cancer Checklist lung cancer biomarker template—anaplastic lymphoma kinase (ALK). Abbreviations: EML4, echinoderm microtubule-associated protein-like 4; KIF5B, kinesin family member 5B; KLC1, kinesin light chain 1; TFG, tropomyosin receptor kinase–fused gene.

Figure 4. (not shown) Examples of tumor biomarker dashboards. Abbreviations: ALK, anaplastic lymphoma kinase; ROS1, ROS proto-oncogene 1, receptor tyrosine kinase.

This system would allow for more efficient, more accurate, and safer methods of providing data for optimizing patient care, with all of the discrete data transmitted electronically and linked to the original tumor report. In Ontario, Canada, this vision is rapidly advancing, as demonstrated by the Cancer Care Ontario successes with eCC implementation and current plans to implement the eCC biomarker templates across the province. Future challenges include the identity and tracking of related tumor samples over time and integration of testing from different laboratories. Because testing on a given specimen can be performed at different times and in different laboratories, a future standard must address the annotation of results with tumor source, procurement dates, and other biospecimen-specific data.30 The relationship of test results from multiple specimens from the same patient needs to be recorded in a standard format so that this parent-child hierarchical relationship can be analyzed over time.

Pathologists are increasingly asked to provide biomarker information for patient care, tumor registries, epidemiologic studies, translational research, and quality improvement activities.20 The eCC model provides a pathway to meet these demands, with efficient and error-free data entry, reporting, and transmission of data elements, and with the ability to produce output that is human readable, efficient to use, and easy to interpret. As the CCPs and eCCs have matured, Ontario pathologists and cancer registries have demonstrated success with large-scale implementations. However, continued improvements are needed. As the field of pathology grows, particularly in the area of biomarkers, structured electronic reporting will become critical to helping physicians provide optimal patient care and will facilitate secondary uses of pathology data.

  1. Robb JA, Gulley ML, Fitzgibbons PL, et al. A call to standardize preanalytic data elements for biospecimens. Arch Pathol Lab Med. 2014; 138(4):526–537.

Molecular Genetic Biomarkers in Myeloid Malignancies
Matynia AP, Szankasi P, Shen W, Kelley TW.
Arch Pathol Lab Med. 2015;139:594–601
http://dx.doi.org:/10.5858/arpa.2014-0096-RA

Recent studies using massively parallel sequencing technologies, so-called next-generation sequencing, have uncovered numerous recurrent, single-gene variants or mutations across the spectrum of myeloid malignancies. Objectives.—To review the recent advances in the understanding of the molecular basis of myeloid neoplasms, including their significance for diagnostic and prognostic purposes and the possible implications for the development of novel therapeutic strategies. Data Sources.—Literature review. Conclusions.—The recurrent mutations found in myeloid malignancies fall into distinct functional categories.

These include (1) cell signaling factors, (2) transcription factors, (3) regulators of the cell cycle, (4) regulators of DNA methylation, (5) regulators of histone modification, (6) RNA-splicing factors, and (7) components of the cohesin complex. As the clinical significance of these mutations and mutation combinations is established, testing for their presence is likely to become a routine part of the diagnostic workup. This review will attempt to establish a framework for understanding these mutations in the context of myeloproliferative neoplasms, myelodysplastic syndromes, and acute myeloid leukemia.

Pathways Affected by Recurrent Mutations in Myeloid Malignancies

Cell Signaling The ability of a cell to respond to diverse physiologic stimuli, including cytokines, chemokines, growth factors, and hormones, or to the presence of bacteria and other microorganisms is mediated via the interaction of specific ligands and their corresponding cell surface receptors. Ligand binding usually results in receptor dimerization and activation of a tyrosine kinase, either intrinsically present in the cytoplasmic domain or as an associated polypeptide. Further propagation of the signal from the cell surface to the nucleus involves the formation of macromolecular complexes and the activation or inactivation of various enzymes. The final outcome of the signal transduction is modulation of the expression of certain genes and their products, which ultimately produces a cellular response. In normal cells, this process is tightly regulated owing to the involvement of negative or inhibitory signals. In tumor cells these processes may be perturbed owing to mutations that impart inappropriate activation or deactivation of enzymatic function. Genes for receptor protein tyrosine kinases, such as FLT3 and KIT, or receptor-associated kinases, such as JAK2, are the most commonly mutated cell-signaling factors in myeloid malignancies. Activating mutations in these proteins occur in narrowly defined hotspots, resulting in ligand-independent dimerization or constitutive kinase activation. An example is the protein tyrosine kinase JAK2, which transduces signals from ligand-bound cell surface receptors for thrombopoietin (TPOR/MPL) and erythropoietin (EPOR).

Activating mutations in JAK2 are commonly found in the myeloproliferative neoplasms (MPNs): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These disorders appear to be driven, in large part, by the inappropriate activation of growth factor– signaling pathways, and JAK2 is central to signaling from EPOR and TPOR/MPL via STAT5, STAT3, the RAS-MAP kinase pathway, and the PI-3 kinase–AKT pathway. Many negative regulators of cytokine signaling, such as SH2B adaptor protein 3 (SH2B3, also known as LNK)1,2 and cytokine-inducible SH2-containing protein (CISH, also known as SOCS)3 keep the pathway in balance. Downstream RAS signaling is counteracted by NF1, a protein that stimulates the intrinsic RAS guanosine triphosphatase activity. An effect similar to JAK2 mutations may be achieved through mutations in other proteins involved in these pathways, including activating mutations of a surface receptor (MPL) or loss-of-function mutations in negative regulators (SH2B3, CISH), all of which promote survival, proliferation, and differentiation of committed myeloid progenitors.4 Example genes included in this group: JAK2, MPL, KIT, FLT3, CSF3R, PTPN11, KRAS, NRAS, and NF1.

Transcription Transcription is a tightly regulated process that depends on the formation and assembly of protein and protein-DNA complexes. These complexes, called transcription factors, bind to specific DNA sequences adjacent to the genes they regulate and promote (in the case of an activator) or block (in the case of a repressor) the recruitment of RNA polymerases to those genes. This regulatory activity controls the formation of messenger RNA (mRNA) transcripts. Mutations that block the activity of transcriptional activators may, in certain circumstances, lead to a block in cellular differentiation due to the lack of the necessary gene products. Many of the transcription factors that are recurrently mutated in myeloid malignancies, such as RUNX1, GATA1, GATA2, and CEBPA, are involved in fundamental aspects of myelopoiesis and it is believed that these mutations lead to a block in myeloid differentiation.  Example genes included in this group: RUNX1, CEBPA, GATA1, GATA2, ETV6, and PHF6.

Epigenetic Modifiers: Regulation of DNA Methylation DNA methylation involves the addition of a methyl group to cytosine bases in the context of cytosine-guanine sequences (so-called CpG sites), leading to the creation of 5-methylcytosine. CpG islands are usually located in or near promoter regions. Their methylation is an important epigenetic mechanism for regulating gene expression and, in the context of heritable methylation patterns, underlies the process of genomic imprinting. Additionally, it has been hypothesized that aberrant DNA methylation may contribute to the pathogenesis of cancers,5–8 including myeloid neoplasms. Although cancer genomes tend to be globally hypomethylated, in comparison with normal tissues, hypermethylation of specific CpG islands at tumor suppressor genes, resulting in their inactivation, is common in many tumors.9

ormation of compact, inactive heterochromatin.10 Several factors regulate the process of DNA methylation. Mutations in some of these factors have been found recurrently in myeloid neoplasms.11–13 DNA methyltransferases catalyze the methylation at the 50 position of cytosine. DNMT3A and DNMT3B are involved in de novo methylation, whereas DNMT1 maintains hemimethylated DNA during replication. Once created, 5-methylcytosine can be further modified by a group of methylcytosine dioxygenases (Ten Eleven Translocation dioxygenases: TET1, TET2, and TET3) to 50-hydroxymethylcytosine, a presumed short-lived intermediary that may lead to demethylation of cytosine.

Mutations in both DNMT3A and TET2 likely lead to loss of function of the respective enzyme activities. Recently, mutations in IDH1 and IDH2 have been identified in myeloid neoplasms and other cancers. Interestingly, recurring mutations of an arginine residue in the active site (R132 in IDH1 and R140 in IDH2) prevent the normal catalytic function of the enzyme (conversion of isocitrate to aketoglutarate) and appear to induce a neomorphic enzyme activity resulting in the formation of the rare oncometabolite 2-hydroxyglutarate.14 TET2 belongs to a family of dioxygenases that requires a-ketoglutarate as a cofactor.15,16 It has been shown that 2-hyroxyglutarate acts as a competitive inhibitor of a-ketoglutarate–dependent dioxygenases, which include TET2 and members of the KDM family of histone demethylases, thereby inducing epigenetic changes at both the level of DNA methylation and histone modification.14,17

Therapeutic inhibitors of mutant forms of IDH proteins and the resulting 2-hydroxyglutarate are also under investigation.18 Example genes included in this group: DNMT3A, TET2, IDH1, and IDH2.

Epigenetic Modifiers: Mutations Affecting Histone Function  Histone proteins are involved in the dynamic organization of DNA into zones of active euchromatin and inactive heterochromatin in a process that is regulated, in part, by a complex series of posttranslational modifications to histone tails, including acetylation and methylation. These modifications affect the recruitment of regulatory proteins such as transcription factors, corepressors, and coactivators, as well as histone-modifying enzymes themselves. Trimethylation of the lysine at position 27 in histone H3 (H3K27), one of the more common modifications, generally leads to reduced gene expression and it would be expected that mutations that reduce methylation at H3K27 would activate transcription. Recently, recurrent mutations in several genes encoding histone regulators, including ASXL1, EZH2, SUZ12, and KDM6A (also known as UTX), have been identified.

Perturbations in epigenetic pathways result in global, genome-wide effects and it is often difficult to identify which altered cellular function eventually leads to neoplasia. The same also holds true for perturbations in the RNA splicing machinery and the cohesion complex (see below). Example genes included in this group: ASXL1, EZH2, SUZ12, and KDM6A.

Cohesin Complex Genes  The cohesin complex is a conserved multimeric protein complex that regulates cohesion of sister chromatids during cell division,21 postreplicative DNA repair,22,23 and global gene expression.24–28
Example genes included in this group: STAG2, RAD21, SMC1A, and SMC3.

RNA-Splicing Factors  RNA splicing results in the formation of mature mRNA transcripts derived from exons, the protein coding portion of the genome. Splicing occurs in a macromolecular complex of small nuclear RNAs and proteins assembled de novo on each pre-mRNA strand in a multistep process. This complex is known as the spliceosome. Transcripts may undergo alternative splicing in a tissue, or context-specific manner and the protein products of alternatively spliced transcripts may have altered function.
Example genes included in this group: SF3B1, SRSF2, ZRSR2, and U2AF1.

Cell Cycle Regulators  Example genes included in this group: TP53 and NPM1.

Genetic Biomarkers in Myeloid Malignancies

Myeloproliferative Neoplasms  Myeloproliferative neoplasms encompass a group of clonal stem cell disorders characterized by expansion of 1 or more of the myeloid lineages resulting in bone marrow hypercellularity and increased peripheral blood myeloid cell counts. The MPN category includes chronic myelogenous leukemia, PV, ET, PMF, chronic neutrophilic leukemia, mastocytosis, and others. The underlying genetic landscape of some of these disorders is very well understood, as in the case of chronic myelogenous leukemia with t(9;22), but is much less well understood in many other entities.

The discovery of a recurrent codon 617 activating mutation (V617F) in exon 14 of the tyrosine kinase JAK237–40 and additional mutations in JAK2 exon 1241 provided the first genetic evidence of the importance of dysregulated growth factor signaling in these disorders. The prevalence of JAK2 mutations in classical MPNs varies from 95% to 99% in PV, 50% to 70% in ET, 40% to 50% in PMF,37–41,43 and molecular tests for their detection are available and widely used in clinical practice. Similarly, activating mutations in the MPL gene, encoding the thrombopoietin receptor, are present in approximately 4% of ET cases and approximately 11% of PMF cases.44–47 Recently, calreticulin (CALR), encoding an endoplasmic reticulum chaperone, has also been shown to be important. Somatic CALR mutations are found in 70% to 84% of patients with ET or PMF with wild-type JAK2 and MPL, 8% of MDS cases, and occasionally in other myeloid neoplasms.48 Clonal analyses suggest CALR mutations act as an initiating mutation in some patients.48 In ET and PMF, CALR mutations and JAK2 and MPL mutations are mutually exclusive,49 and CALR mutations appear to be absent in PV.49 CALR mutations appear to be primarily insertion or deletion mutations that result in a frameshift and the subsequent generation of a novel C-terminal peptide.48,49

Many other genes involved in intracellular signaling, such as negative regulators of the JAK2 signaling pathway, are mutated in MPNs. Among these is SH2B3, which negatively regulates JAK2 activation through its SH2 domain. Mutations in SH2B3 during the chronic phase are uncommon, fewer than 5% in ET and PMF50; however, their frequency increases during leukemic transformation, suggesting a role in disease progression.51 Another negative regulator found mutated in MPNs is the Cbl proto-oncogene, E3 ubiquitin protein ligase gene (CBL). CBL acts as a multifunctional adapter with ubiquitin ligase activity and by competitive blockade of signaling.

A shift from a simple, chronic myelogenous leukemia–like model for MPN pathophysiology to a more complex model occurred with the emergence of evidence of a ‘‘pre-JAK2’’ genetic event. This concept is based on the observation that mutations in signaling molecules are not sufficient for disease development and that several cooperating genetic hits appear to be required.4 Mutations in genes involved in epigenetic regulation, including EZH2, ASXL1, and TET2 (also found in many other myeloid neoplasms), are postulated to act as those initialing events, preceding JAK2V617F mutations.55 EZH2 mutations do not occur in ET, but are present in 3% of PVs and 13% of MFs.56 ASXL1 mutations are found in only approximately 7% of patients with ET and PV but more frequently in PMF cases (from 19%–40%).57,58 TET2 mutations occur in approximately 14% of MPNs, ranging from 11% in ETs to 19% in PMFs.59,60 Finally, there are mutations that are rarely found during the chronic phase but which may be present at transformation, and are therefore thought to play a role in disease progression. IDH1 and IDH2 mutations, for example, have a low frequency in the chronic phase (0.8% in ET, 1.9% in PV, and 4.2% in MF) but a much higher frequency in blast phase.61

Myelodysplastic Syndromes and MDS/MPN Overlap Disorders 

The myelodysplastic syndromes are a group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, morphologic evidence of dysplasia in at least 1 of the myeloid lineages, peripheral cytopenias, bone marrow hypercellularity, and an increased risk of development of AML.74 Clonal cytogenetic abnormalities, including large deletions and chromosome gains, as well as balanced translocations, are observed in approximately 50% of MDS cases by routine methods,74 and their identification aids in establishing the diagnosis and may provide prognostic information.

Acute Myelogenous Leukemia  Acute myeloid leukemia is a genetically heterogeneous disease resulting in the accumulation of myeloblasts in bone marrow with a concomitant reduction in normal hematopoiesis. The diagnosis and subclassification of AML depends on detecting the presence of recurrent cytogenetic abnormalities.74 In many cases, particularly those that are cytogenetically normal (CN-AML), several single-gene mutations further aid in the stratification of disease outcomes. The significance of mutations in genes such as FLT3, NPM1, and CEBPA is well established but nextgeneration sequencing has led to the discovery of numerous additional recurrent mutations, including in TET2,12,59 ASXL1,104 IDH1/IDH2,13,105,106 DNMT3A,11,107 and PHF6.108

Among the most common mutations found in de novo AML are NMP1, FLT3, and DNMT3A mutations, present in 22% to 29%, 22% to 37%, and 15% to 26% of samples, respectively.31,110,111 Other genes less commonly targeted by mutations are IDH1/IDH2 (15%– 20%), KRAS/NRAS (12% combined), RUNX1 (5%–10%),TET2 (8%–14%), TP53 (2%–8%), CEBPA (6%–14%), WT1 (6%–8%), PTPN11 (4%), and KIT (4%–6%).31,110,111

The prognostic significance of a subset of recurrent mutations is well established. In CN-AML, biallelic CEBPA mutations127,128 and NPM1 mutations without FLT3-ITD mutations129–133 are associated with a favorable prognosis. In contrast, FLT3-ITD without NPM1 mutations112,113,129–132 and MLL–partial tandem duplication mutations134–136 portend poor outcome. KIT mutations in AML with t(8;21) or inv(16)131,137 are also associated with unfavorable outcome. The European LeukemiaNet panel138 first proposed a standardized classification according to both cytogenetic and molecular genetic data to allow a better comparison of prognosis among patients with AML. However, only mutations of NPM1, CEBPA, and FLT3 were included in their recommendations. The relevance of more recently discovered mutations, including IDH1, IDH2, WT1, TET2, ASXL1, among others, remains unclear.139–142 The presence of certain mutations may also allow for more targeted therapeutic regimens; for example, FLT kinase inhibitors may be useful in cases with mutations and IDH1 inhibitors are under investigation in patients with IDH1 mutations.143,144

An enormous amount of new information illuminating the genetic complexity of myeloid neoplasms has been generated during the last few years. Much work remains to be done but it is clear that the future role of the pathologist in collecting and interpreting this information will be an essential component of the management of these patients.

 

The Cancer Genomics Resource List 2014
Zutter MM, Bloom KJ, Cheng L, Hagemann IS, et al.
Arch Pathol Lab Med. http://dx.doi.org:/10.5858/arpa.2014-0330-C

Optimizing the Clinical Utility of Biomarkers in Oncology: The NCCN Biomarkers Compendium
Marian L. Birkeland, Joan S. McClure
Arch Pathol Lab Med. 2015;139:608–611
http://dx.doi.org:/10.5858/arpa.2014-0146-RA

The rapid development of commercial biomarker tests for oncology indications has led to confusion about which tests are clinically indicated for oncology care. By consolidating biomarker testing information recommended within National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (NCCN Guidelines), the NCCN Biomarkers Compendium aims to ensure that patients have access to appropriate biomarker testing based on the evaluations and recommendations of the expert NCCN panel members.
Objectives.—To present the recently launched NCCN Biomarkers Compendium. Data Sources.—Biomarker testing information recommended within NCCN Clinical Treatment Guidelines as well as published resources for genetic and biological information. Conclusions.—The NCCN Biomarkers Compendium is a continuously updated resource for clinicians who need access to relevant and succinct information about biomarker testing in oncology and is linked directly to the recommendations provided within the NCCN Clinical Practice Guidelines.

Most recommendations contained within the NCCN Guidelines are based upon lower-level evidence and uniform NCCN consensus (category 2A).1 This is not a deficiency of the guidelines, but is rather because high-level evidence is not available for most decisions across the continuum of care. A deeper look at what constitutes a ‘‘recommendation’’ might begin to clarify that issue. A recommendation can include all of the recommended workup, surgical options, options for chemotherapy, and tests recommended for ongoing surveillance. Although many of these options are routinely used as standard of care in clinical practice, there is often not the available body of high-level evidence that supports category 1 recommendations, thus most are category 2A levels of evidence and consensus. In other instances, recommendations for chemotherapy regimens for which there is high-level, randomized clinical trial evidence are listed as category 1.

Several derivative products arise from the NCCN Guidelines. The NCCN Drugs & Biologics Compendium (NCCN Compendium) is a resource outlining appropriate use of drugs and biologics as recommended in the NCCN Guidelines. To be included in the compendium, an agent must first be recommended in at least 1 of the NCCN Guidelines. The compendium is typically used by clinicians and payors to determine appropriate use and as a standard to determine coverage. The rapid development and commercialization of biomarker and companion diagnostic testing in cancer gave rise to the NCCN Biomarkers Compendium, to be used by both payors and clinicians to facilitate identification of biomarker tests recommended for use by NCCN guideline panels. The NCCN uses a broad definition of ‘‘biomarker’’ for the purposes of this compendium. All tests measuring genes or gene products, which are used for diagnosis, screening, monitoring, surveillance, or for providing predictive or prognostic information, are included in the biomarkers compendium. This compendium focuses on the biology of the biomarker itself and its clinical utility in supporting clinical decision making. Information is organized by the biomarker being measured, and not by listing of commercially available tests or test kits. Close to 1000 biomarker testing recommendations are included in the NCCN Biomarkers Compendium.

The NCCN Biomarkers Compendium is presented on the NCCN Web site as a series of drop-down menus, allowing users to pick from menus listing Guideline, Disease, Molecular Abnormality, or Gene Symbol.3 Users can retrieve all recommendations for a particular disease, or can select a gene-based search in order to show which diseases have a validated use for a particular gene test. Additional fields can be displayed by selecting from a series of boxes to the right of the drop-down menus (see Figure 1, which shows default fields for RAS testing in colon cancer). Once records are displayed, the resulting table can be sorted by the information in any of the displayed columns. If a searcher chooses, all records can be displayed and then searched with any text term or sorted by any of the columns for a more comprehensive picture of the contents of the database.

Figure 1. (not shown)  KRAS mutation testing recommendation from the National Comprehensive Cancer Network (NCCN) Biomarkers Compendium.3 Reproduced with permission from the NCCN Biomarkers Compendium [1] 2014 National Comprehensive Cancer Network, Inc. (NCCN.org; accessed February 21, 2014). To view the most recent and complete version of the NCCN Biomarkers Compendium, go online to NCCN.org. National Comprehensive Cancer Network, NCCN, NCCN Guidelines, and all other NCCN content are trademarks owned by the National Comprehensive Cancer Network, Inc.

Disease Description Colon cancer
Specific Indication Metastatic disease
Molecular Abnormality KRAS/NKRAS mutation
Test KRAS/NKRAS
Chromosome 1p13.2, 12p12.1
Gene Symbol KRAS/NKRAS
Test Detects Mutation
Methodology
Category of Evidence 2A
Specimen Types FFPE tumor tissue
Recommendation …Determination of RAS mutations.
Test Purpose Predictive
Guideline Page COL 4 of 5, COL 4, COL 9
Note All patients with metastatic colorectal cancer should be genotyped for RAS mutations. At the very least …

Figure 2. (Table)  Example of PDF file generated from ‘‘print’’ command of National Comprehensive Cancer Network (NCCN) Biomarkers Compendium record. Reproduced with permission from the NCCN Biomarkers Compendium [1] 2014 National Comprehensive Cancer Network, Inc (NCCN.org; accessed February 21, 2014). To view the most recent and complete version of the NCCN Biomarkers Compendium, go online to NCCN.org. National Comprehensive Cancer Network, NCCN, NCCN Guidelines, and all other NCCN content are trademarks owned by the National Comprehensive Cancer Network, Inc.

Table 2. (List) Summary of Testing Types Included in the National Comprehensive Cancer Network Biomarkers Compendiuma,b

Protein expression
Translocation
Mutation
Chromosomal defect
Gene rearrangement
Virus detection
Antigen expression
Serum proteins
Amplification
Short repeated sequences
Promoter methylation
Gene expression pattern
Helicobacter pylori

Table 3. Predictive Tests Used for Treatment Decision Making, Extracted From National Comprehensive Cancer Network Guidelines and Biomarkers Compendiuma,b

Test   Disease
21-gene RT-PCR

BCR-ABL1 translocation

Breast cancer
ABL1 mutation Ph+ acute lymphoblastic leukemia, chronic myelogenous leukemia
ALK rearrangement Non–small cell lung cancer
BRAF mutation Non–small cell lung cancer, melanoma, colon cancer, rectal cancer
EGFR mutation Non–small cell lung cancer
ERBB2 amplification/overexpression Breast cancer, esophageal and esophagogastric junction cancers, gastric cancer
ESR1 expression Breast cancer
KIT mutation Soft tissue sarcoma: GIST
KRAS mutation Colon cancer, rectal cancer, non–small cell lung cancer
MGMT promoter methylation Central nervous system cancers: anaplastic glioma/glioblastoma
MLH1, MSH2, MSH6, PMS2 expression and/or mutation, MSI testing Colon cancer, rectal cancer
PDGFRA mutation Soft tissue sarcoma: GIST
PGR expression Breast cancer
ROS1 rearrangement Non–small cell lung cancer

A large number of tests were grouped for the purposes of this simplified table into the category of gross chromosomal abnormalities. Interestingly, the guidelines so far contain only a single recommendation for the use of a gene expression profiling test, and this is the 21-gene reverse transcription–polymerase chain reaction test recommended within the breast cancer treatment guideline, where the score for this test can be used as part of a decision-making process for chemotherapy recommendations in node negative, hormone receptor–positive, HER2-negative disease.

Table 3 summarizes the biomarker tests included in the NCCN Biomarkers Compendium that are predictive for either responsiveness (eg, BRAF mutation and vemurafenib sensitivity) or nonresponsiveness (eg, KRAS mutation testing and cetuximab or panitumumab insensitivity) to a particular type of therapy. As the number of companion diagnostics and targeted therapies grows, we expect this category of test to become one of the largest categories of testing contained within the Biomarkers Compendium, and it may be surprising to note that only 15 of these types of test are currently recommended within the NCCN Guidelines.

The NCCN Biomarkers Compendium generally avoids recommendations for particular methodologies or test kits to use to assess mutations and translocations. The choice of methodology and supplier for carrying out the recommended biomarker tests remains that of the treating oncologists and pathologists.

The NCCN Biomarkers Compendium may be used by payers as a reference for coverage decisions and by clinicians as a guide to which biomarkers are appropriate to test. The Biomarkers Compendium focuses on the clinical usefulness of biomarker testing rather than specific tests or test kits that identify the presence or absence of the marker. Other groups are continuing to assess clinical and analytic validity for specific biomarker test methodologies. Even the US Food and Drug Administration approval process is limited to clinical and analytic validity, and does not specifically address clinical utility. The NCCN Biomarkers Compendium is complementary to these other efforts. By providing biomarker testing information, the NCCN Biomarkers Compendium aims to ensure that patients have coverage and access to appropriate biomarker testing, based on the evaluations and recommendations of the expert NCCN panel members.

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Gastrointestinal Endocrinology

Writer and Curator: Larry H Bernstein, MD, FCAP

The Gut Microbial Endocrine Organ: Bacterially Derived Signals Driving Cardiometabolic DiseasesMark Brown and Stanley L. Hazen

Annual Review of Medicine Jan 2015; 66: 343-359
http://dx.doi.org:/10.1146/annurev-med-060513-093205

The human gastrointestinal tract is home to trillions of bacteria, which vastly outnumber host cells in the body. Although generally overlooked in the field of endocrinology, gut microbial symbionts organize to form a key endocrine organ that converts nutritional cues from the environment into hormone-like signals that impact both normal physiology and chronic disease in the human host. Recent evidence suggests that several gut microbial-derived products are sensed by dedicated host receptor systems to alter cardiovascular disease (CVD) progression. In fact, gut microbial metabolism of dietary components results in the production of proatherogenic circulating factors that act through a meta-organismal endocrine axis to impact CVD risk. Whether pharmacological interventions at the level of the gut microbial endocrine organ will reduce CVD risk is a key new question in the field of cardiovascular medicine. Here we discuss the opportunities and challenges that lie ahead in targeting meta-organismal endocrinology for CVD prevention.

Exogenous glucagon-like peptide 1 reduces contractions in human colon circular muscle

Antonella Amato, Sara Baldassano, Rosa Liotta1, Rosa Serio and Flavia Mulè
J Endocrinol April 1, 2014 221 29-37
http://dx.doi.org:/10.1530/JOE-13-0525

Glucagon-like peptide 1 (GLP1) is a naturally occurring peptide secreted by intestinal L-cells. Though its primary function is to serve as an incretin, GLP1 reduces gastrointestinal motility. However, only a handful of animal studies have specifically evaluated the influence of GLP1 on colonic motility. Consequently, the aims of this study were to investigate the effects induced by exogenous GLP1, to analyze the mechanism of action, and to verify the presence of GLP1 receptors (GLP1Rs) in human colon circular muscular strips. Organ bath technique, RT-PCR, western blotting, and immunofluorescence were used. In human colon, exogenous GLP1 reduced, in a concentration-dependent manner, the amplitude of the spontaneous contractions without affecting the frequency and the resting basal tone. This inhibitory effect was significantly reduced by exendin (9–39), a GLP1R antagonist, which per se significantly increased the spontaneous mechanical activity. Moreover, it was abolished by tetrodotoxin, a neural blocker, or Nω-nitro-L-arginine – a blocker of neuronal nitric oxide synthase (nNOS). The biomolecular analysis revealed a genic and protein expression of the GLP1R in the human colon. The double-labeling experiments with anti-neurofilament or anti-nNOS showed, for the first time, that immunoreactivity for the GLP1R was expressed in nitrergic neurons of the myenteric plexus. In conclusion, the results of this study suggest that GLP1R is expressed in the human colon and, once activated by exogenous GLP1, mediates an inhibitory effect on large intestine motility through NO neural release.

The impact of dipeptidyl peptidase 4 inhibition on incretin effect, glucose tolerance, and gastrointestinal-mediated glucose disposal in healthy subjects

N A Rhee, S H Østoft, J J Holst, C F Deacon, T Vilsbøll and F K Knop
Eur J Endocrinol September 1, 2014 171 353-36
http://dx.doi.org:/10.1530/EJE-14-0314

Objective Inhibition of dipeptidyl peptidase 4 (DPP4) is thought to intensify the physiological effects of the incretin hormones. We investigated the effects of DPP4 inhibition on plasma levels of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP1), incretin effect, glucose tolerance, gastrointestinal-mediated glucose disposal (GIGD) and gastric emptying in healthy subjects. Design A randomised, controlled and open-labelled study. Methods Ten healthy subjects (six women; age, 40±5 years (mean±S.E.M.); BMI, 24±3 kg/m2; fasting plasma glucose, 5.1±0.2 mmol/l and HbA1c, 34±1 mmol/mol (5.3±0.1%)) were randomised to two-paired study days comprising a 4-h 50 g oral glucose tolerance test (OGTT) with paracetamol (A) and an isoglycaemic intravenous (i.v.) glucose infusion (B), with (A1+B1) and without (A2+B2) preceding administration of the DPP4 inhibitor sitagliptin. Results Isoglycaemia was obtained in all subjects on the paired study days. Significant increases in fasting levels and OGTT-induced responses of active GLP1 and GIP were seen after DPP4 inhibition. No significant impact of DPP4 inhibition on fasting plasma glucose (5.1±0.1 vs 4.9±0.1 mmol/l, P=0.3), glucose tolerance (area under the curve (AUC) for plasma glucose, 151±35 vs 137±26 mmol/l×min, P=0.7) or peak plasma glucose during OGTT (8.5±0.4 vs 8.1±0.3 mmol/l, P=0.3) was observed. Neither incretin effect (40±9% (without DPP4 inhibitor) vs 40±7% (with DPP4 inhibitor), P=1.0), glucagon responses (1395±165 vs 1223±195 pmol/l×min, P=0.41), GIGD (52±4 vs 56±5%, P=0.40) nor gastric emptying (Tmax for plasma paracetamol: 86±9 vs 80±12 min, P=0.60) changed following DPP4 inhibition. Conclusions These results suggest that acute increases in active incretin hormone levels do not affect glucose tolerance, GIGD, incretin effect, glucagon responses or gastric emptying in healthy subjects.

Morphology and Tissue Distribution of Four Kinds of Endocrine Cells in the Digestive Tract of the Chinese Yellow Quail (Coturnix japonica)

He, M., Liang, X., Wang, K., (…), Li, X., Liu, L.
Analytical and Quantitative Cytology and Histology 2014; 36 (4), pp. 199-205

Objective: To describe the tissue distribution, density, and the morphological characteristics of 4 kinds of endocrine cells in the digestive tract of the Chinese yellow quail (Coturnix japonica). Study design: The streptavidin-biotin-peroxidase complex immunohistochemical method was used to identify the distribution of somatostatin (SS), serotonin (5-HT), gastrin and neuropeptide Y (NPY) in digestive tracts including proventriculus, duodenum, jejunum, ileum, and rectum. SPSS 19.0 software was used to perform biological statistical analysis. Results: The results showed that the SS and 5-HT secreting cells were mainly distributed in the proventriculus (19.2±6.9 and 16.1±3.4 cfu/mm2) and duodenum (2.9±2.0 and 1.9±0.6 cfu/mm2). Gastrin and NPY were not detected in each section of the digestive tract. Moreover, there was no significant difference in the quantitative distribution and morphological characteristics of SS and 5-HT secreting cells in the digestive tract between male and female quails. Conclusion: The distribution and morphological characteristics of endocrine cells were closely related to the physiological functions of different parts in the digestive tract. The preferential location of endocrine cells provides additional information for future studies on the physiological roles of gastrointestinal peptides in the gastrointestinal tract of the Chinese yellow quail

GEP-NETS update: Functional localisation and scintigraphy in neuroendocrine tumours of the gastrointestinal tract and pancreas (GEP-NETs)

Wouter W de Herder
Eur J Endocrinol May 1, 2014 170 R173-R183
http://dx.doi.org:/10.1530/EJE-14-0077

For patients with neuroendocrine tumours (NETs) of the gastrointestinal tract and pancreas (GEP) (GEP-NETs), excellent care should ideally be provided by a multidisciplinary team of skilled health care professionals. In these patients, a combination of nuclear medicine imaging and conventional radiological imaging techniques is usually mandatory for primary tumour visualisation, tumour staging and evaluation of treatment. In specific cases, as in patients with occult insulinomas, sampling procedures can provide a clue as to where to localise the insulin-hypersecreting pancreatic NETs. Recent developments in these fields have led to an increase in the detection rate of primary GEP-NETs and their metastatic deposits. Radiopharmaceuticals targeted at specific tumour cell properties and processes can be used to provide sensitive and specific whole-body imaging. Functional imaging also allows for patient selection for receptor-based therapies and prediction of the efficacy of such therapies. Positron emission tomography/computed tomography (CT) and single-photon emission CT/CT are used to map functional images with anatomical localisations. As a result, tumour imaging and tumour follow-up strategies can be optimised for every individual GEP-NET patient. In some cases, functional imaging might give indications with regard to future tumour behaviour and prognosis.

An immunohistochemical study on the distribution of endocrine cells in the digestive tract of gray goose (Anser anser)

Jun YANG1, Lei ZHANG,, Xin LI, , Leii ZHANG, , Xiangjiang LIU, , Kemei PENG

Turk. J. Vet. Anim. Sci. 2012; 36(4): 373-379
http://dx.doi.org:/10.3906/vet-1101-654

The objective of this study was to investigate the morphology and the distribution of 5-hydroxytryptamine (5-HT), somatostatin (SS), gastrin (Gas), glucagon (Glu), and substance P immunoreactive (IR) cells in the digestive tract of gray goose by the immunohistochemical streptavidin-peroxidase method.

The samples were taken from 10 healthy  adult gray geese. Th e results showed that 5 kinds of IR cells were mainly distributed between the mucous epithelium and intestinal gland. The number of 5-HT-IR cells was highest in the rectum and duodenum, but none were observed  in the pylorus. SS-IR cells appeared in great numbers in the pylorus, duodenum, and cecum; however, they were not found in esophagus. Gas-IR cells were mainly distributed in the glandular stomach and jejunum. Glu-IR cells appeared  in small numbers in the glandular stomach, duodenum, and jejunum, but were not detected in other tissues. Substance  P-IR cells were located in the jejunum, cecum, and rectum. Analysis of the present study showed that the distribution and morphological features of these 5 different endocrine cells were related to the feeding habits and metabolism in the digestive tract of the gray goose

Chapter 154 – Somatostatin

Mathias Guggera, Jean-Claude Meunierb

Handbook of Biologically Active Peptides 2006, Pages 1123–1130
http:/dx.doi.org:/10.1016/B978-012369442-3/50157-4

Somatostatin is abundant in the mucosa and in the enteric nervous system of the gastrointestinal tract and in the pancreas. In these tissues, it exerts a broad range of mainly inhibitory physiological actions in multiple targets, including endocrine glands, exocrine glands, smooth muscles, blood vessels, and immune cells, mediated by up to six somatostatin receptor subtypes. Several diseases of the gastrointestinal tract are characterized by disturbances in the somatostatin production or by overexpression of somatostatin receptors. In particular, somatostatin receptors have been found to be overexpressed in neuroendocrine gastroenteropancreatic tumors. These tumors can be diagnostically and therapeutically targeted with somatostatin analogs. In addition, various nonneoplastic diseases, including bleeding in the upper gastrointestinal tract, fistulas, and diarrhea can also be treated with somatostatin analogs.

Immunocytochemical study of the distribution of endocrine cells in the pancreas of the Brazilian sparrow species Zonotrichia Capensis Subtorquata (Swaison, 1837)

Nascimento, AA.*; Sales, A.; Cardoso, TRD.; Pinheiro, NL.; Mendes, RMM.
Braz. J. Biol. Nov. 2007; 67(4):  São Carlos

In the present study, we investigated types of pancreatic endocrine cells and its respective peptides in the Brazilian sparrow species using immunocytochemistry. The use of polyclonal specific antisera for somatostatin, glucagon, avian pancreatic polypeptide (APP), YY polypeptide (PYY) and insulin, revealed a diversified distribution in the pancreas. All these types of immunoreactive cells were observed in the pancreas with different amounts. Insulin- Immunoreactive cells to (B cells) were most numerous, preferably occupying the central place in the pancreatic islets. Somatostatin, PPA, PYY and glucagon immunoreactive cells occurred in a lower frequency in the periphery of pancreatic islets.

Immunolocalisation of the serotonin in the fundus ventriculi and duodenum of the Asia minor ground squirrel: (Spermophilus xanthoprymnus)

Timurkaan, S., Özkan, E., Ilgün, R., Gür, F.M
Veterinarski Arhiv 2009; 79 (1), pp. 69-76

Serotonin immunoreactive cells were located and distributed in the fundus and duodenum with variable frequencies. They were spherical or spindle-shaped and the highest frequency serotonin immunoreactive cells were detected in the whole fundic region. The regional distribution of the endocrine cells in the fundus and duodenum of the citellus resembled other mammalian species.

An Immunohistochemical Study of Gastrointestinal Endocrine Cells in the BALB/c Mouse

Ku, S.K., Lee, H.S., Lee, J.H.
J Vet Med Series C: Anatomia Histologia Embryologia 2004; 33 (1), pp. 42-48

The distributions and frequencies of some endocrine cells in the eight portions of the gastrointestinal tract (GIT) of BALB/c mouse were studied. Endocrine cells were stained using immunohistochemical method with seven types of anti-sera against bovine chromogranin (BCG), serotonin, gastrin, cholecystokinin (CCK)-8, somatostatin, glucagon and human pancreatic polypeptide (HPP), and the regional distributions and their relative frequencies were observed in the eight portions of the GIT of BALB/c mice. All seven types of immunoreactive (IR) cells were identified. Most of the IR cells in the intestinal portion were generally spherical or spindle in shape (open type cell) while round-shaped cells (closed type cell) were found in the intestinal gland and stomach regions occasionally. Their relative frequencies varied according to each portion of the GIT. BCG-IR cells were observed throughout the whole GIT except for the rectum and they were most predominant in the pylorus. Serotonin-IR cells were detected throughout the whole GIT and they showed the highest frequency in the fundus. Gastrin- and CCK-IR cells were restricted to the pylorus and duodenum with a majority in the pylorus and rare or a few frequencies in the duodenum. Compared with other mammals, somatostatin-IR cells were restricted to the fundus and pylorus with a few frequencies, respectively. In addition, glucagon- and HPP-IR cells were restricted to the fundus and duodenum, respectively, with relative low frequencies. Some species-dependent unique distributions and frequencies of endocrine cells were observed in the GIT of BALB/c mouse compared with other rodents.

Immunohistochemical study of the distribution of serotonin in the gastrointestinal tract of the porcupines (Hystrix cristata)

Timurkaan, S., Karan, M., Aydin, A.
Revue de Medecine Veterinaire 2005; 156 (11), pp. 533-536

Serotonin immunoreactive cells were located in the gastric glands and in the intestinal epithelium with variable frequencies. They were spherical or spindle-shaped. Serotonin immuno-reactive cells were detected in almost all regions of the gastrointestinal tract and they showed highest frequency in the stomach and colon.

Effects of carbachol on gastrin and somatostatin release in rat antral tissue culture

Wolfe, M.M., Jain, D.K., Reel, G.M., McGuigan, J.E.
Gastroenterology 1984; 87 (1), pp. 86-93

Recent studies have demonstrated that somatostatin-containing cells are in close anatomic proximity to gastrin-producing cells in antral mucosa, suggesting a potential local regulatory role for somatostatin. The purpose of this study was to examine further the relationships between gastrin and somatostatin and the effects of the cholinergic agonist carbachol on content and release of gastrin and somatostatin using rat antral mucosa in tissue culture. Antral mucosa was cultured at 37 °C in KrebsHenseleit buffer, pH 7.4, gassed with 95% O2-5% CO2. After 1 h, the culture medium was decanted and the tissue was boiled to extract mucosal gastrin and somatostatin. Inclusion of carbachol 2.5 × 10-6 M in the culture medium decreased medium somatostatin from 1.91 ± 0.28 (SEM) ng/mg tissue protein to 0.62 ± 0.12 ng/mg (p < 0.01), extracted mucosal somatostatin from 2.60 ± 0.30 to 1.52 ± 0.16 ng/mg (p < 0.001), and percentage of somatostatin released from 42% ± 2.6% to 27% ± 2.2% (p < 0.01). Carbachol also increased culture media gastrin from 14 ± 2.5 to 27 ± 3.0 ng/mg protein (p < 0.01). Tissue content and release of gastrin and somatostatin were also examined during culture of rat antral mucosa in culture media containing antibodies to somatostatin in the presence and in the absence of carbachol. Incubation with somatostatin antisera, both with and without carbachol, markedly increased culture media concentrations of somatostatin, all of which was effectively bound by antibodies present in the media. Antibody binding of somatostatin was accompanied by significant increases in culture media gastrin concentrations, both in the presence and in the absence of carbachol. Results of these studies support the hypothesis that antral somatostatin exerts a local regulatory effect on gastrin release and that cholinergic stimulation of gastrin release is mediated, at least in part, through inhibition of somatostatin synthesis and release.

Endogenous somatostatin-28 modulates postprandial insulin secretion. Immunoneutralization studies in baboons

J W Ensinck, R E Vogel, E C Laschansky, D J Koerker, et al.
J Clin Invest. 1997; 100(9):2295–2302.
http://dx.doi.org:/10.1172/JCI119767

Somatostatin-28 (S-28), secreted into the circulation from enterocytes after food, and S-14, released mainly from gastric and pancreatic D cells and enteric neurons, inhibit peripheral cellular functions. We hypothesized that S-28 is a humoral regulator of pancreatic B cell function during nutrient absorption. Consistent with this postulate, we observed in baboons a two to threefold increase in portal and peripheral levels of S-28 after meals, with minimal changes in S-14. We attempted to demonstrate a hormonal effect of these peptides by measuring their concentrations before and after infusing a somatostatin-specific monoclonal antibody (mAb) into baboons and comparing glucose, insulin, and glucagon-like peptide-1 levels before and for 4 h after intragastric nutrients during a control study and on 2 d after mAb administration (days 1 and 2). Basal growth hormone (GH) and glucagon levels and parameters of insulin and glucose kinetics were also measured. During immunoneutralization, we found that (a) postprandial insulin levels were elevated on days 1 and 2; (b) GH levels rose immediately and were sustained for 28 h, while glucagon fell; (c) basal insulin levels were unchanged on day 1 but were increased two to threefold on day 2, coincident with decreased insulin sensitivity; and (d) plasma glucose concentrations were similar to control values. We attribute the eventual rise in fasting levels of insulin to its enhanced secretion in compensation for the heightened insulin resistance from increased GH action. Based on the elevated postmeal insulin levels after mAb administration, we conclude that S-28 participates in the enteroinsular axis as a decretin to regulate postprandial insulin secretion.

The Therapeutic Value of Somatostatin and Its Analogues

Sadaf Farooqi, John S. Bevan, Michael C. Shepperd, John A. H. Wass
Pituitary June 1999; 2(1), pp 79-88
http:/dx.doi.org:/10.1023/A:1009978106476

In this review we discuss the physiological effects of somatostatin, which are mediated by specific receptor subtypes on different tissues. These observations have suggested new therapeutic possibilities for the use of the synthetic somatostatin analogues in the treatment of acromegaly as well as a number of other endocrine and non-endocrine disorders.

Somatostatin and Somatostatin Receptors

Ujendra Kumar, Michael Grant
Cellular Peptide Hormone Synthesis and Secretory Pathways
(Results and Problems in Cell Differentiation) 2010; 50: pp 97-120
http://dx.doi.org:/10.1007/400_2009_29

The biological effects of somatostatin (SST) were first encountered unexpectedly in the late 1960s in two unrelated studies, one by Krulich et al. (1968) who reported on a growth hormone (GH)-releasing inhibitory substance from hypothalamic extracts, and the other, by Hellman and Lernmark (1969), on the presence of a potent insulin inhibitory factor from the extracts of pigeon pancreatic islets. However, the inhibitory substance was not officially identified until 1973 by Guillemin’s group (Brazeau et al. 1973). In both synthetic and naturally occurring forms, this tetradecapeptide, originally coined as somatotropin release-inhibitory factor (SRIF, SST-14) was shown by Brazeau et al. to be the substance controlling hypothalamic GH release. This single achievement not only pioneered SST research but was also duly recognized, as Guillemin shared the 1977 Nobel Prize in Medicine. The following years bequeathed an exponential increase in SST-related studies. It soon became clear that SST-synthesis was not restricted to the hypothalamus. Its production is widely distributed throughout the central nervous system (CNS), peripheral neurons, the gastrointestinal tract, and the pancreatic islets of Langerhans (Luft et al. 1974; Arimura et al. 1975; Dubois 1975; Hokfelt et al. 1975; Orci et al. 1975; Pelletier et al. 1975; Polak et al. 1975; Patel and Reichlin 1978). In fact, SST-like immunoreactivity can be found throughout various tissues of vertebrates and invertebrates, including the plant kingdom (Patel 1992; Tostivint et al. 2004). Given its broad anatomical distribution, it is no wonder that SST produces a wide spectrum of biological effects. Generally regarded as an inhibitory factor, SST can function either locally on neighboring cells or distantly through the circulation, to regulate such physiological processes as glandular secretion, neurotransmission, smooth muscle contractility, nutrient absorption, and cell division (Reichlin 1983a, b; Patel 1992, 1999; Patel et al. 2001; Barnett 2003).

Receptor-Mediated Tumor Targeting with Radiopeptides. Part 1. General Concepts and Methods: Applications to Somatostatin Receptor-Expressing Tumors

Alex N. Eberle, Gabriele Mild, and Sylvie Froidevaux
Journal of Receptors and Signal Transduction  2004; 24(4) , Pages 319-455
http://dx.doi.org:/10.1081/RRS-200040939

Radiolabeled peptides have become important tools in nuclear oncology, both as diagnostics and more recently also as therapeutics. They represent a distinct sector of the molecular targeting approach, which in many areas of therapy will implement the old “magic bullet” concept by specifically directing the therapeutic agent to the site of action. In this three-part review, we present a comprehensive overview of the literature on receptor-mediated tumor targeting with the different radiopeptides currently studied. Part I summarizes the general concepts and methods of targeting, the selection of radioisotopes, chelators, and the criteria of peptide ligand development. Then, the >400 studies on the application to somatostatin/somatostatin-release inhibiting factor receptor-mediated tumor localization and treatment will be reviewed, demonstrating that peptide radiopharmaceuticals have gained an important position in clinical medicine.

The somatostatin neuroendocrine system: physiology and clinical relevance in gastrointestinal and pancreatic disorders

Malcolm J. Low
Best Practice & Res Clin Endocr & Metab, 2004; 18(4), pp. 607–622
http://dx.doi.org:/10.1016/j.beem.2004.08.005

The physiologic functions of hypothalamic somatostatin in the regulation of pituitary hormone secretion and the clinical use of somatostatin analogs for the treatment of pituitary adenomas have been reviewed. Similarly, the distribution, normal function and potential pathogenic roles of somatostatin in the central nervous system have been reported in detail. This review will focus exclusively on the physiologic actions of somatostatin and its receptors in the gastrointestinal tract, pancreas and immune system. Diagnostic and therapeutic roles of somatostatin analogs in a diverse catalog of neoplastic, inflammatory and autoimmune conditions affecting peripheral systems are outlined, with an emphasis on both well-established indications and current areas of exploration.

Somatostatin is produced in enteroendocrine D cells and intrinsic neurons of the stomach, intestines and pancreas. Its physiologic actions are mediated primarily by somatostatin receptors type 2 and 5, and include the inhibition of secretion of most endocrine and exocrine factors. Diseases directly attributable to somatostatin excess or deficiency are rare, although there is a complex pathogenic relationship between persistent Helicobacter pylori infection and reduced somatostatin in chronic gastritis. Abundant somatostatin receptors on many neoplastic and inflammatory cells are the basis for sensitive in vivo imaging with radiolabeled somatostatin analogs and provide a therapeutic target. Current indications for somatostatin therapy include hormone-expressing neuroendocrine tumors, intractable diarrhea and variceal bleeding secondary to portal hypertension. Exciting advances are being made in the development of high-affinity nonpeptide analogs with receptor-subtype selectivity and increased bioavailability. Somatostatin analogs coupled to high-energy radionuclides show promise as novel cytotoxic agents for certain metastatic tumors.

Evolution of the somatostatin gene family Both forms of mammalian somatostatin are derived post-translationally from a common pro-hormone by the action of specific pro-protein convertases (PCs). Genetic studies indicate a primary role for PC2 in the generation of SST147, which is the predominant form of somatostatin produced in the brain and most other tissues. SST28 is found in its highest concentrations in the gastrointestinal tract, especially the mucosal epithelial cells of the intestines.
A revised evolutionary concept of the somatostatin gene family is that a primordial gene underwent duplication during or before the advent of chordates and that the two resulting genes subsequently underwent differing rates of mutation to produce the distinct prepro-somatostatin and prepro-cortistatin genes in mammals. A second gene duplication event likely occurred in teleosts to generate PSS1 and PSS-II from the ancestral somatostatin gene.
It is possible that additional related genes have not yet been identified. Recent studies utilizing unique polyclonal antisera and a strain of somatostatin-deficient mouse have demonstrated the existence of a novel gastrointestinal peptide with homology to the amino acid sequence of SST28(1–13) that has been named thrittene.
Somatostatin gene organization and regulation The mammalian PPS1 (or SMST) gene has a relatively simple organization consisting of two coding exons separated by one intron. A single promoter directs transcription of the PPS1 gene in all tissues, and there are no known alternative mRNA splicing events. The molecular mechanisms underlying the developmental and hormonal regulation of somatostatin gene transcription have been most extensively studied in pancreatic islets and islet-derived cell lines. The proximal enhancer elements in the somatostatin gene promoter that bind complexes of homeodomain-containing transcription factors (PAX6, PBX, PREP1) to upregulate transcription in pancreatic islets may actually represent gene silencer elements in neurons (promoter elements TSEII and UE-A). Conversely, another related cis-element in the somatostatin gene (promoter element TSEI) apparently binds a homeodomain transcription factor PDX1 (also called STF1/ IDX1/IPF1) that is common to developing brain, pancreas and foregut, and regulates gene expression in both the CNS and gut.
Enteroendocrine cells of the gut mucosa differentiate from pluripotential stem cells in the crypts, share molecular phenotypes and retain close paracrine interactions among the daughter cells. Similarly, pancreatic islet cells share common precursors. Recent studies have demonstrated that bone marrow contains a stem cell population capable of producing islet-like cell clusters in vitro that contain somatostatin-positive cells together with the other cell types found in normally differentiated islets.
Somatostatin Receptors  There are five somatostatin receptor subtypes (SSTR1–5) encoded by separate genes located on different chromosomes. Alternative mRNA splicing generates SSTR2α and SSTR2β from heteronuclear RNA after transcription from the single SSTR2 gene. SSTRs are members of the rhodopsin-like G protein-coupled receptor superfamily and are most closely related structurally to the opioid receptors. The unique amino acid signature of SSTRs is contained in a seven-element fingerprint of peptide sequences located in conserved regions of the N and C termini, extra- and intra-cellular loops, and transmembrane domains. SSTRs are expressed in discrete or partially overlapping distributions in multiple target organs and differ in their coupling to second messenger signaling molecules, and therefore in their range and mechanism of intracellular actions. The subtypes also differ in their binding affinity to specific somatostatin-like ligands. Some of these differences have important implications for the use of somatostatin analogs in diagnostic imaging and in pharmacotherapy.
All SSTR subtypes are coupled to pertussis toxin-sensitive G proteins and bind SST14 and SST28 with high affinity in the low nanomolar range, although SST28 has a modestly higher affinity for SSTR5. All the subtypes are expressed in brain and pituitary to varying degrees with different distributions, but SSTR2 and SSTR5 are clearly the most abundant in peripheral tissues. These two subtypes are also the most physiologically important in pancreatic islets. SSTR5 is responsible for the inhibition of insulin secretion from b-cells, and SSTR2 is essential for the inhibition of glucagon from a-cells. SSTR1 is expressed at low levels in gastrointestinal structures. The binding of somatostatin to its receptors leads to the activation of one or more inhibitory G proteins (Gi/o), which in turn decrease adenylyl cyclase activity and the concentration of intracellular cAMP. Other G protein-mediated actions common to all The somatostatin neuroendocrine system 609 SSTRs are activation of a vanadate-sensitive phosphotyrosine phosphatase (PTP) and modulation of mitogen-activated protein kinases (MAPKs).
Inhibition of endocrine and exocrine secretion Somatostatin has diverse biologic activities in the gastrointestinal system. It is secreted from D cells into the extracellular space to act as a paracrine factor on nearby endocrine cells and as an autocrine factor to inhibit its own secretion. Most of the circulating hormonal somatostatin originates from the stomach and intestines. Basal plasma levels are in the range of 30–100 pg/ml and increase postprandially by as much as 100% over baseline for a duration up to 2 hours. The release of somatostatin from enteric D cells is regulated by a combination of nutritional, humoral, neural and paracrine signals.

The modulatory role of somatostatin in gastric acid secretion by parietal cells illustrates the typical complexity of hormonal, paracrine and neural integration within the gastrointestinal tract.
Somatostatin secreted from gastric D cells modulates the gastrin-enterochromaffin-like cell—parietal cell axis. Gastrin, secreted from G cells, stimulates the release of histamine from enterochromaffin-like cells (ECL), which is in turn a major secretagog of hydrochloric acid (HCl) from gastric parietal (P) cells. Somatostatin (SST14) inhibits secretion from each of these cell types, although the predominant actions are on the G and ECL cells. Food intake mediates gastric acid secretion by activating both vagal nerves and intrinsic gastric neurons. D cells are stimulated by the autocrine release of amylin, the paracrine release of bombesin and atrial natiuretic peptide (ANP), the enteric neuron release of pituitary adenylate cyclase-activating peptide (PACAP) and cholecystokinin (CCK), and the T lymphocyte release of interleukin-4 (IL-4).D cells are inhibited by histamine acting on H3 receptors in a negative paracrine feedback loop from ECL cells and by other factors, including gamma-aminobutyric acid (GABA) and opioid peptides. The pathways illustrated are not all-inclusive but represent many of the key regulatory steps.

Practice points

† long-acting somatostatin analogs are primary therapeutic tools for the symptomatic treatment of the excessive hormone and monoamine secretion from carcinoids and other neuroendocrine tumors

† somatostatin and long-acting somatostatin analogs are effective first-line
medical treatment for upper gastrointestinal bleeding from esophageal varices associated with hepatic cirrhosis and portal hypertension but are not indicatedfor the treatment of bleeding from gastric varices or duodenal ulcers

† radiolabeled somatostatin analogs provide a sensitive imaging technique for a wide range of neoplastic and inflammatory disorders, including neuroendocrine tumors, meningiomas and sarcoidosis because of their high level expression of somatostatin receptors.

The role(s) of somatostatin, structurally related peptides and somatostatin receptors in the gastrointestinal tract: a review

J Van Op den bosch, D Adriaensen, L Van Nassauw, Jean-Pierre Timmermans
Regulatory Peptides 156 (2009) 1–8
http://dx.doi.org:/10.1016/j.regpep.2009.04.003

Extensive functional and morphological research has demonstrated the pivotal role of somatostatin (SOM) in the regulation of a wide variety of gastrointestinal activities. In addition to its profound inhibitory effects on gastrointestinal motility and exocrine and endocrine secretion processes along the entire gastrointestinal tract, SOM modulates several organ-specific activities. In contrast to these well-known SOM-dependent effects, knowledge on the SOM receptors (SSTR) involved in these effects is much less conclusive. Experimental data on the identities of the SSTRs, although species- and tissue-dependent, point towards the involvement of multiple receptor subtypes in the vast majority of gastrointestinal SOM-mediated effects. Recent evidence demonstrating the role of SOM in intestinal pathologies has extended the interest of gastrointestinal research in this peptide even further. More specifically, SOM is supposed to suppress intestinal inflammatory responses by interfering with the extensive bidirectional communication between mucosal mast cells and neurons. This way, SOM not only acts as a powerful inhibitor of the inflammatory cascade at the site of inflammation, but exerts a profound anti-nociceptive effect through the modulation of extrinsic afferent nerve fibers. The combination of these physiological and pathological activities opens up new opportunities to explore the potential of stable SOM analogues in the treatment of GI inflammatory pathologies.

Schematic overview of the distribution of the SSTRs 1–5

Schematic overview of the distribution of the SSTRs 1–5

Schematic overview of the distribution of the SSTRs 1–5 in the murine small intestine under control conditions (left panel) and during intestinal schistosomiasis (right panel). In non-inflamed conditions, SSTR1, SSTR2A and SSTR4 are expressed in non-neuronal (glial cells, enterocytes…) and neuronal cells, both from intrinsic and extrinsic origin. SSTR3 and SSTR5 are undetectable. In response to intestinal schistosomiasis, profound sprouting of nerve fibres expressing SSTR1, SSTR3 and SSTR4 is observed, in addition to the expression of SSTR1 and SSTR3 in mucosal mast cells (MMC).

Somatostatin and Its Receptor Family

Yogesh C. Patel
Frontiers in Neuroendocrinology 1999; 20, 157–198 Article ID frne.1999.0183

Somatostatin (SST), a regulatory peptide, is produced by neuroendocrine, inflammatory, and immune cells in response to ions, nutrients, neuropeptides, neurotransmitters, thyroid and steroid hormones, growth factors, and cytokines. The peptide is released in large amounts from storage pools of secretory cells, or in small amounts from activated immune and inflammatory cells, and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells that are widely distributed in the brain and periphery. These actions are mediated by a family of seven  transmembrane (TM) domain G-protein-coupled receptors that comprise five distinct subtypes (termed SSTR1–5) that are endoded by separate genes segregated on different chromosomes. The five receptor subtypes bind the natural SST peptides, SST-14 and SST-28, with low nanomolar affinity. Short synthetic octapeptide and hexapeptide analogs bind well to only three of the subtypes, 2, 3, and 5. Selective nonpeptide agonists with nanomolar affinity have been developed for four of the subtypes (SSTR1, 2, 3, and 4) and putative peptide antagonists for SSTR2 and SSTR5 have been identified. The ligand binding domain for SST ligands is made up of residues in TMs III–VII with a potential contribution by the second extracellular loop. SSTRs are widely expressed in many tissues, frequently as multiple subtypes that coexist in the same cell. The five receptors share common signaling pathways such as the inhibition of adenylyl cyclase, activation of phosphotyrosine phosphatase (PTP), and modulation of mitogen-activated protein kinase (MAPK) through G-protein-dependent mechanisms.

Somatostatin receptors

Lars Neisig Møller, Carsten Enggaard Stidsen, Bolette Hartmann, Jens Juul Holst
Biochimica et Biophysica Acta 1616 (2003) 1 – 84
http://dx.doi.org:/10.1016/S0005-2736(03)00235-9

In 1972, Brazeau et al. isolated somatostatin (somatotropin release-inhibiting factor, SRIF), a cyclic polypeptide with two biologically active isoforms (SRIF-14 and SRIF-28). This event prompted the successful quest for SRIF receptors. Then, nearly a quarter of a century later, it was announced that a neuropeptide, to be named cortistatin (CST), had been cloned, bearing strong resemblance to SRIF. Evidence of special CST receptors never emerged, however. CST rather competed with both SRIF isoforms for specific receptor binding. And binding to the known subtypes with affinities in the nanomolar range, it has therefore been acknowledged to be a third endogenous ligand at SRIF receptors. This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing the characteristic seven-transmembrane-segment (STMS) topography. Years of intensive research have resulted in cloning of five receptor subtypes (sst1-sst5), one of which is represented by two splice variants (sst2A and sst2B). The individual subtypes, functionally coupled to the effectors of signal transduction, are differentially expressed throughout the mammalian organism, with corresponding differences in physiological impact. It is evident that receptor function, from a physiological point of view, cannot simply be reduced to the accumulated operations of individual receptors. Far from being isolated functional units, receptors co-operate. The total receptor apparatus of individual cell types is composed of different-ligand receptors (e.g. SRIF and non-SRIF receptors) and co-expressed receptor subtypes (e.g. sst2 and sst5 receptors) in characteristic proportions. In other words, levels of individual receptor subtypes are highly cell-specific and vary with the co-expression of different-ligand receptors. However, the question is how to quantify the relative contributions of individual receptor subtypes to the integration of transduced signals, ultimately the result of collective receptor activity. The generation of knock-out (KO) mice, intended as a means to define the contributions made by individual receptor subtypes, necessarily marks but an approximation. Furthermore, we must now take into account the stunning complexity of receptor co-operation indicated by the observation of receptor homo- and heterodimerisation, let alone oligomerisation. Theoretically, this phenomenon adds a novel series of functional megareceptors/super-receptors, with varied pharmacological profiles, to the catalogue of monomeric receptor subtypes isolated and cloned in the past. SRIF analogues include both peptides and non-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype-selective analogues. Several have become available.

Somatostatin And Its Analogues In The Therapy Of Gastrointestinal Disease

Wynick, J. M. Polak And S. R. Bloom
Pharmac. Ther. 1989; 41, pp. 353-370

During the course of efforts to determine the distribution of growth hormone-releasing factor (GHRF) in rat hypothalamus a substance that inhibited growth hormone release was unexpectedly detected by Krulich et aL (1968). Their findings led them to hypothesize that the secretion of growth hormone from the pituitary was regulated by two different interacting neurohumoral factors–one stimulatory, the other inhibitory–each under the control of the nervous system. At about the same time Hellman and Lernmark (1969) found a factor in extracts of pigeon pancreatic islet-cells that inhibited insulin release in vivo from cultured pancreatic islet-cells. These two observations, seemingly unrelated, were ultimately to converge with the chemical identification of somatostatin, as an inhibitory peptide found in both the hypothalamus and pancreas.

Growth hormone-release inhibitory activity was re-discovered in 1972 by Brazeau et al. (1973). A concentrated effort to isolate and sequence the active principal was successful and it proved to be a cyclic peptide, to which the term ‘somatostatin’ (somatotrophin release inhibitory factor) was applied.               Subsequent work (Reichlin, 1982a,b, 1983a,b; Iverson, 1983; Guillemin, 1978a,b) has considerably expanded the initially simple concept of somatostatin as a 14 amino-acid containing peptide (tetradecapeptide), bridged by a sulphur-sulphur bond whose main function was the regulation of growth-hormone secretion (Bonfils, 1985). Somatostatin related peptides are now known to constitute a family that includes the original identified peptide (designated somatostatin 14), an N-terminal extended somatostatin (somatostatin 28), several species specific variants and larger prohormone forms.
The name somatostatin may now be considered to be inappropriate because this compound is distributed widely in cells that have nothing to do with growth-hormone regulation or release. Tissues where somatostatin may be found include the nervous system, the gut and endocrine glands.
Somatostatin is present in every vertebrate class and even in primitive invertebrates (Vale et al., 1976; Falkmer et al., 1978; Jackson, 1978). This would suggest that this molecule and its controlling gene or genes evolved before the appearance on earth of differentiated cell-cell and nerve-cell communication (Roth et al., 1982). The evolutionary paths of mammals and fish are thought to have diverged at least 400 million years ago. The fact that the phenotype of somatostatin 14 is so well conserved (as to a lesser degree is that of somatostatin 28) suggests that throughout evolutionary history the specific configuration of somatostatin 14 has endowed a selective advantage on the animal kingdom, and its absence is not compatible with life.
Though widely distributed in cells throughout the body of vertebrates somatostatin does not in Guillemin’s words (1978a), “inhibit secretion of everything and anything” (since, for example it has no effect on the release of LH and FSH). Despite this it has certainly earned itself the nickname ‘endocrine cyanide’ (Bloom and Polak, 1987). The peptide is found in most but not all organs and displays specific and selective functions depending on its location. Within the nervous system somatostatinergic neurons are found in the cortex, limbic system, anterior pituitary, brain stem and spinal cord.
The various biological effects of somatostatin seem to be mediated through its specific high affinity receptors found in the brain, pituitary, adrenal, pancreas and gastrointestinal tract. Not only normal target tissue, but also tumors from the same endocrine tissues i.e. human pituitary adenomas, human and hamster pancreatic insulinomas, glucagonomas and VIPomas all bear somatostatin receptors (Reubi et al., 1981, 1982a, 1984a, 1985a, 1987a,b). Interestingly, tumors from tissues which are not established targets for somatostatin also seem to bear somatostatin receptors (Goodman et al., 1982; Reubi et al., 1986). Reubi et al. (1987b) demonstrated that many endocrine tumors including meningiomas, breast, pancreatic and pituitary tumors all have somatostatin receptors however, they demonstrated no receptors in prostatic carcinomas, ovarian carcinomas, endometrial carcinomas, primary liver cell carcinomas, pheochromocytomas, aldosterone secreting tumors, medullary carcinoma of the thyroid and a number of pulmonary carcinoids. Somatostatin receptors were also found in benign or malignant tumors originating from tissues not primarily known as somatostatin target organs, the biological function of such receptors is therefore unknown though it may be that they mediate the anti-proliferative effect of somatostatin and may therefore potentially be of therapeutic interest (Blankenstein et al., 1983, 1984).

Review article: somatostatin analogues in the treatment of gastroenteropancreatic neuroendocrine (carcinoid) tumoursModlin,

M. Pavel[1], M. Kidd & B. I. Gustafsson
Aliment Pharmacol Ther 2009; 31, 169–188
http://dx.doi.org:/10.1111/j.1365-2036.2009.04174.x

Background

The discovery of somatostatin (SST) and the synthesis of a variety of analogues constituted a major therapeutic advance in the treatment of gastroenteropancreatic neuroendocrine (carcinoid) tumours (GEP-NETs). They currently provide the most efficient treatment to achieve symptomatic relief and have recently been demonstrated to inhibit tumour growth.

Aim To review 35 years of experience regarding the clinical application and

efficacy of SST analogues. Methods The PubMed database (1972–2009) was searched using somatostatin as a search term with combinations of terms including ‘treatment’; ‘neuroendocrine’; ‘carcinoid’; ‘tumor’; ‘octreotide’; ‘lanreotide’ and ‘pasireotide’. Results In a review of 15 studies including 481 patients, the slow-release formulations Sandostatin LAR and Somatuline SR⁄ Autogel achieved symptomatic relief in 74.2% (61.9–92.8%) and 67.5% (40.0–100%), biochemical response in 51.4% (31.5–100%) and 39.0% (17.9–58%), and tumor response in 69.8% (47.0–87.5%) and 64.4% (48.0–87.0%) respectively. New SST analogues like SOM230 (pasireotide) that exhibit pan SST receptor activity and analogues with high affinity to specific somatostatin receptor (sstr) subtypes show promise. Conclusion As more precise understanding of NET cell biology evolves and molecular biological tools advance, more accurate identification of individual tumours sstr profile will probably facilitate a more precise delineation of SST analogue treatment.

Novel Autonomic Neurotransmitters And Intestinal Function

S. Taylor and R. A. R. Bywater
Pharmac. Ther. 1989; 40, pp. 401 to 438

In this review we will discuss some of the difficulties encountered in ascribing a neurotransmitter function to the more recently discovered peptides and other substances within the intestine. We will also provide a brief (and of necessity incomplete) account of some of the properties of intestinal putative neurotransmitters, and their possible roles in the functions of the small and large intestine.
The Enteric Nervous System The diverse intestinal functions associated with transit, digestion and absorption rely upon an intact enteric nervous system. The enteric nervous system essentially consists of those neurons whose cell bodies lie within the walls of the gastrointestinal tract. In the small and large intestine the cell bodies lie within the myenteric and submucous plexuses; their processes ramify throughout the majority of the intestinal wall and in many areas give rise to additional plexuses (Furness and Costa, 1987; Gabella, 1987). Functionally, these neurons can be divided into sensory neurons, interneurons and motor neurons. Some enteric neurons receive projections from extrinsic neurons and/or send projections centrally; we will not consider these projections further here.
The early observations of the co-existence of peptides in the enteric nervous system (Schultzberg et al., 1980) have now been extended and these studies demonstrate that the co-existence of two or more peptides is the rule rather than the exception (H6kfelt et al., 1987). The mix of peptides within neurons does not appear to be random; rather, there appears to be a systematic grouping of peptides in neurons with particular projections. This has led to the concept of “chemical coding” of enteric neurons. According to this concept, particular combinations of peptides are associated with particular neural pathways and perhaps with particular functions. For example, in the guinea pig small intestine, two chemically coded groups of submucous neurons have projections of different lengths running to the mucosa. Cell bodies with longer projections show immunoreactivity for dynorphin (DYN) and VIP. The other group shows immunoreactivity for choline acetyltransferase (CHAT), cholecystokinin, (CCK), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and somatostatin (SOM) (Costa et al., 1986a; Furness et al., 1987a). More recently it has been demonstrated that both groups of neurons show immunoreactivity for galanin (GAL) (Furness et al., 1987a,b). As for the neurotransmitter roles in the gut, the key question then becomes; “How does the presence of specific combinations of chemical substances (including peptides) relate to neuronal function?” It has been known for several years that “classical” transmitter substances can coexist in combination with various peptides (H6kfelt et al., 1980; Gilbert and Emson, 1983).
The above commentary upon the possible co-existence of several putative transmitter substances highlights the complex neurochemistry of the enteric nervous system. A corresponding degree of complexity appears to exist for the neuronal circuitry that ultimately directs the differing, but highly organized, patterns of motility and the secretory/absorptive functions of the intestinal tract. In vitro electrophysiological studies of the myenteric and submucous plexuses have indicated that several different types of neurons are present, each with their own biophysical characteristics. Furthermore, neurotransmission through, and probably between, the plexuses involves synaptic potentials which have time courses ranging from several milliseconds up to several minutes, depending upon the characteristics (stimulus strength, frequency and train length, etc.) and location of the applied electrical stimulus (see Wood, 1987, for references). Intracellular recordings from smooth muscle cells have also shown that excitatory and inhibitory junction potentials (EJPs and IJPS) of varying time courses can be evoked at various locations along the intestine during transmural electrical stimulation in response to selective stimulus regimens (see, for instance, Bywater and Taylor, 1986).
A number of authors have proposed criteria which should be fulfilled in order that transmitter status can be bestowed upon a particular substance (see Furness and Costa, 1982, for references). These criteria were developed with reference to the classical transmitter substances such as ACh, using the paradigm of a single transmitter per neuron. Regardless of the coexistence of several putative transmitters, status can only be granted to those substances that are found to be released from that nerve terminal. In the enteric nervous system a particular putative transmitter may be contained in several different functional pathways. However, in general, the methods used for eliciting release of putative transmitter substances (e.g. transmural electrical stimulation) are not specific for particular projections. Thus, for any substance, the association of demonstrated release with a given transmitter role is not facile.

New roles of the multidimensional adipokine: Chemerin

Syeda Sadia Fatima, Rehana Rehman, Mukhtiar Baig, Taseer Ahmed Khan
Peptides 62 (2014) 15–20
http://dx.doi.org/10.1016/j.peptides.2014.09.019

The discovery of several adipokines with diverse activities and their involvement in regulation of various pathophysiological functions of human body has challenged the researchers. In the family of adipokine, chemerin is a novel and unique addition. Ever since the first report on chemerin as a chemo-attractant protein, there are numerous studies showing a multitasking capacity of chemerin in the maintenance of homeostasis, for the activation of natural killer cells, macrophages and dendritic cells in both innate and adaptive immunity. Its diversity ranges from generalized inflammatory cascades to being explicitly involved in the manifestation of arthritis, psoriasis and peritonitis. Its association with certain cancerous tissue may render it as a potential tumor marker. In present review, we aim to consolidate recent data of investigations on chemerin in context to functional characteristics with a special reference to its role as a metabolic signal in inflammation and non-metabolic syndromes.

Neuropeptide Y is expressed in subpopulations of insulin- and non-insulin-producing islet cells in the rat after dexamethasone treatment: a combined immunocytochemical and in situ hybridisation study

Myrs6n a, *, B. Ahr6n b, F. Sundler
Regulatory Peptides 1995; 60, 19-31

Neuropeptide Y (NPY) is known to occur in adrenergic and non-adrenergic nerves in rat pancreatic islets. Analysis of islet extracts has revealed local NPY synthesis after glucocorticoid treatment. The cellular localization of NPY expression in rat islets following dexamethasone treatment (2 mg/kg daily, for 12 days), was investigated by a combination of immunocytochemistry (ICC) and in situ hybridization (ISH). NPY-immunoreactive nerve fibers were seen in pancreatic islets of both control and dexamethasone-treated rats. In the controls weak NPY immunoreactivity but no NPY mRNA was observed in occasional i:dets. After dexamethasone treatment, clusters of islet cells distributed both centrally and peripherally displayed intense NPY immunoreactivity and NPY mRNA labelling. Immunocytochemical double staining and ISH combined with ICC for NPY and islet hormones revealed that most NPY expressing cells were identical with insulin cells; a few cells were identical[ with somatostatin or pancreatic polypeptide (PP) cells. In contrast, glucagon cells seemed to be devoid of NPY immunoreactivity and NPY mRNA labelling. Thus, in the rat, glucocorticoids cause a marked upregulation of NPY expression in islet cells, preferentially the insulin cells. The expression of NPY might represent an islet adaptation mechanism to the reduced peripheral insulin sensitivity.

Neuropeptide Y is expressed in islet somatostatin cells of the hamster pancreas: a combined immunocytochemical and in situ hybridization study

Ulrika Myrsrn, Frank Sundler
Regulatory Peptides 1995; 57, 65-76

Neuropeptide Y (NPY) is known to occur in the autonomic nervous system, including the pancreatic islet innervation. We now present evidence that NPY is also expressed in endocrine islet cells in hamster pancreas. Thus, NPY-immunoreactivity and gene expression were detected in peripheral islet cells, using immunocytochemistry (ICC), in situ hybridization (ISH), and a combination of these techniques. Double immunostaining for NPY and somatostatin enabled localization of NPY to the vast majority of the somatostatin cells. However, a few somatostatin cells were devoid of NPY immunoreactivity and an occasional NPY-immunoreactive cell was devoid of somatostatin. ISH with an NPY mRNA specific probe, showed labelling of cells in the islet periphery. Furthermore, combined ISH for NPY mRNA and ICC for somatostatin showed autoradiographic labelling of somatostatin cells to a varying degree. Both somatostatin and NPY are inhibitors of insulin and/or glucagon secretion. Thus, in the islets these two peptides may be coreleased and cooperate in the, regulation of islet hormone secretion. The role for NPY emanating from islet cells is probably paracrine rather than endocrine.

Neuropeptide Y and Peptide YY Immunoreactivities in the Pancreas of Various Vertebrates

Wei-Guang Ding, Hiroshi Kimura, Masaki Fujimura And Mineko Fujimiya
Peptides,  1997; 18(10), pp. 1523–1529   PII S0196-9781(97)00237-4

NPY-like immunoreactivity was observed in nerve fibers and endocrine cells
in pancreas of all species examined except the eel, which showed no NPY innervation. The density of NPY-positive nerve fibers was higher in mammals than in the lower vertebrates. These nerve fibers were distributed throughout the parenchyma, and were particularly associated with the pancreatic duct
and vascular walls. In addition, the density of NPY-positive endocrine cells was found to be higher in lower vertebrates than mammals; in descending order; eel 5 turtle 5 chicken . bullfrog . mouse 5 rat 5 human . guinea pig 5 dog. These NPY-positive cells in the eel and certain mammals tended to be localized throughout the islet region, whereas in the turtle and chicken they were mainly scattered in the exocrine region. PYY-immunoreactivity was only present in the pancreatic endocrine cells of all species studied, and localized similarly to NPY. Thus these two peptides may play endocrine or paracrine roles in the regulation of islet hormone secretion in various vertebrate species.

Inhibitory effect of somatostatin on inflammation and nociception

Erika Pintér, Zsuzsanna Helyes, János Szolcsányi
Pharmacology & Therapeutics 112 (2006) 440–456

Somatostatin is released from capsaicin-sensitive, peptidergic sensory nerve endings in response to noxious heat and chemical stimuli such as vanilloids, protons or lipoxygenase products. It reaches distant parts of the body via the circulation and exerts systemic anti-inflammatory and analgesic effects. Somatostatin binds to G-protein coupled membrane receptors (sst1–sst5) and diminishes neurogenic inflammation by prejunctional action on sensory-efferent nerve terminals, as well as by postjunctional mechanisms on target cells. It decreases the release of pro-inflammatory neuropeptides from sensory nerve endings and also acts on receptors of vascular endothelial, inflammatory and immune cells. Analgesic effect is mediated by an inhibitory action on peripheral terminals of nociceptive neurons, since circulating somatostatin cannot exert central action.
Somatostatin itself is not suitable for drug development because of its broad spectrum and short elimination half-life, stable, receptor-selective agonists have been synthesized and investigated. The present overview is aimed at summarizing the physiological importance of somatostatin and sst receptors, pharmacological significance of synthetic agonists and their potential in the development of novel anti-inflammatory and analgesic drugs. These compounds might provide novel perspectives in the pharmacotherapy of acute and chronic painful inflammatory diseases, as well as neuropathic conditions.

the sources, target cells and effects of somatostatin (SST) involved in inflammatory and nociceptive processes

the sources, target cells and effects of somatostatin (SST) involved in inflammatory and nociceptive processes

This schematic drawing demonstrates the sources, target cells and effects of somatostatin (SST) involved in inflammatory and nociceptive processes

Characterization, detection and regulation of somatostatin receptors

The physiological actions of SST are initiated by its binding to membrane receptors. Five human somatostatin receptors (sst), have been cloned and characterized and referred to as sst1-5 receptors using the nomenclature suggested by Hoyer et al. (1995). Structurally, sst receptors are 7 transmembrane domain glycoproteins, comprised of 7 membrane spanning α helical domains connected by short loops, an N-terminal extracellular domain and a C-terminal intracellular domain. On the basis of binding studies using synthetic somatostatin analogs, sst receptors can be divided into 2 different subgroups: SRIF1 group comprising sst2, sst3 and sst5 are able to bind octapeptide analogs, whereas SRIF2 group comprising sst1 and sst4 have negligible affinity for these compounds. Within sst2 receptors, sst2A and sst2B are encoded on the same chromosome 17 and generated through alternative splicing of sst2 mRNA (Patel et al., 1993). None of the peptide analogs bind exclusively to only one of the sst subtypes, although new approaches might yield subtype-selective agonists and antagonists (Hofland et al., 1995; Hoyer et al., 1995; Patel et al., 1995; Reisine & Bell, 1995; Florio & Schettini, 1996; Patel, 1997; Meyerhof, 1998; Janecka et al., 2001). Somatostatin receptors are linked to multiple cellular effector systems via G-proteins. They mediate the inhibition of adenylate cyclase activity (Jakobs et al., 1983; Patel et al., 1995), reduce the conductance of voltage-dependent Ca2+ channels (Schally, 1988; Patel et al., 1995) and activate K+ channels (Mihara et al., 1987; Moore et al., 1988; Wang et al., 1989). Somatostatin receptors also mediate the stimulation of tyrosine phosphatase activity, induce a reduction of cell proliferation and inhibit a Na+/H+ exchanger (NHE1) (Barber et al., 1989; Buscail et al., 1994; Patel et al., 1995). Sst receptors represent a major class of inhibitory receptors which play an important role in modulating higher brain functions, secretory processes, cell proliferation and apoptosis.
Endogenous Somatostatin-28 Modulates Postprandial Insulin Secretion Immunoneutralization Studies in Baboons

John W. Ensinck, Robin E. Vogel, Ellen C. Laschansky, Donna J. Koerker, et al.
J Clin Invest 1997. 100: 2295–2302.).  http://dx.doi.org/10.1172/JCI119767

Somatostatin-28 (S-28), secreted into the circulation from enterocytes after food, and S-14, released mainly from gastric and pancreatic δ cells and enteric neurons, inhibit peripheral cellular functions. We hypothesized that S-28 is a humoral regulator of pancreatic β cell function during nutrient absorption. Consistent with this postulate, we observed in baboons a two to threefold increase in portal and peripheral levels of S-28 after meals, with minimal changes in S-14. We attempted to demonstrate a hormonal effect of these peptides by measuring their concentrations before and after infusing a somatostatin-specific monoclonal antibody (mAb) into baboons and comparing glucose, insulin, and glucagon-like peptide-1 levels before and for 4 h after intragastric nutrients during a control study and on 2 d after mAb administration (days 1 and 2). Basal growth hormone (GH) and glucagon levels and parameters of insulin and glucose kinetics were also measured. During immunoneutralization, we found that
(a) postprandial insulin levels were elevated on days 1 and 2;
(b) GH levels rose immediately and were sustained for 28 h, while glucagon fell; (c) basal insulin levels were unchanged on day 1 but were increased two to threefold on day 2, coincident with decreased insulin sensitivity; and
(d) plasma glucose concentrations were similar to control values.
We attribute the eventual rise in fasting levels of insulin to its enhanced secretion in compensation for the heightened insulin resistance from increased GH action. Based on the elevated postmeal insulin levels after mAb administration, we conclude that S-28 participates in the enteroinsular axis as a decretin to regulate postprandial insulin secretion.

Effects of glucagon-like peptide 1 on appetite and body weight: focus on the CNS

L van Bloemendaal, J S ten Kulve, S E la Fleur, R G Ijzerman and M Diamant
Journal of Endocrinology 2014; 221, T1–T16
http://dx.doi.org:/10.1530/JOE-13-0414

The delivery of nutrients to the gastrointestinal tract after food ingestion activates the secretion of several gut-derived mediators, including the incretin hormone glucagon-like peptide 1 (GLP-1). GLP-1 receptor agonists (GLP-1RA), such as exenatide and liraglutide, are currently employed successfully in the treatment of patients with type 2 diabetes mellitus. GLP-1RA improve glycaemic control and stimulate satiety, leading to reductions in food intake and body weight. Besides gastric distension and peripheral vagal nerve activation, GLP-1RA induce satiety by influencing brain regions involved in the regulation of feeding, and several routes of action have been proposed. This review summarises the evidence for a physiological role of GLP-1 in the central regulation of feeding behavior and the different routes of action involved. Also, we provide an overview of presently available data on pharmacological stimulation of GLP-1 pathways leading to alterations in CNS activity, reductions in food intake and weight loss.

Critical role for peptide YY in protein-mediated satiation and body-weight regulation

Rachel L. Batterham, Helen Heffron, Saloni Kapoor, Joanna E. Chivers, et al.
Cell Metab 2006; 4, 223–233 http://dx.doi.org:/10.1016/j.cmet.2006.08.001

Dietary protein enhances satiety and promotes weight loss, but the mechanisms by which appetite is affected remain unclear. We investigated the role of gut hormones, key regulators of ingestive behavior, in mediating the satiating effects of different macronutrients. In normal-weight and obese human subjects, high-protein intake induced the greatest release of the anorectic hormone peptide YY (PYY) and the most pronounced satiety. Long-term augmentation of dietary protein in mice increased plasma PYY levels, decreased food intake, and reduced adiposity. To directly determine the role of PYY in mediating the satiating effects of protein, we generated PYY null mice, which were selectively resistant to the satiating and weight-reducing effects of protein and developed marked obesity that was reversed by exogenous PYY treatment. Our findings suggest that modulating the release of endogenous satiety factors, such as PYY, through alteration of specific diet constituents could provide a rational therapy for obesity.

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Controlling focused-treatment of Prostate cancer with MRI

Writer and reporter: Dror Nir, PhD.

In recent years there is a growing trend of treating prostate cancer in a way that will preserve, at least partially, the functionality of this organ. When patients are presenting at biopsy a low-grade localized disease, they might be offered focused treatment of the cancer lesion. One of the option is treatment by high-intensity focused ultrasound (HIFU).

The offering of such treatments created the need of controlling their outcome while the prostate is still inside the patient’s body. The most commonly used protocol is following up the patient’s PSA levels and performing “control” biopsies. The biopsies part is at best case; extremely unpleasant. It also bears some risk for complications.

Therefore, urologists are constantly seeking an imaging based protocol that will enable them to assess the treatment outcome without the need for biopsy. The publication I bring below presents the possibility of using MRI for this task. Although it is not recent, it contains many images that makes the story very clear for the reader.  The main weakness of the study is the small number of patients – only 15.

MR Imaging of Prostate after Treatment with High-Intensity Focused Ultrasound

Alexander P. S. Kirkham, FRCR, Mark Emberton, FRCS, Ivan M. Hoh, MRCS, Rowland O. Illing, MRCS, A. Alex Freeman, FRCP and Clare Allen, FRCR

From the Department of Imaging, University College London Hospitals NHS Foundation Trust, England (A.P.S.K., C.A.); Institute of Urology (M.E., I.M.H., R.O.I.) and Department of Histopathology (A.A.F.), University College London, England.

Address correspondence to A.P.S.K., Imaging Department, University College Hospital, 235 Euston Road, London, England NW1 2BU (e-mail: alexkirkham@yahoo.com).

Radiology March 2008; 246 (3) – 833-844.

Abstract

Purpose: To prospectively evaluate magnetic resonance (MR) imaging findings after high-intensity focused ultrasound (HIFU) treatment of the prostate and to correlate them with clinical and histologic findings.

Materials and Methods: Local ethics committee approval and informed consent were obtained. Fifteen consecutive men aged 46–70 years with organ-confined prostate cancer underwent ultrasonographically guided ablation of the whole prostate. Postoperative MR images were obtained within 1 month (12 patients), at 1–3 months (five patients), and in all patients at 6 months. Prostate volume was measured on T2-weighted images, and enhancing tissue was measured on dynamic images after intravenous administration of gadopentetate dimeglumine. Prostate-specific antigen (PSA) level was measured at regular intervals, and transrectal biopsy was performed in each patient at 6 months after treatment.

Results: Initial post-HIFU images showed a central nonenhancing area, surrounded by an enhancing rim. At 6 months, the prostate was small (median volume reduction, 61%) and was of predominantly low signal intensity on T2-weighted images. The volume of prostate enhancing on the initial posttreatment image correlated well with serum PSA level nadir (Spearman r = 0.90, P < .001) and with volume at 6 months (Pearsonr = 0.80, P = .001). The three patients with the highest volume of enhancing prostate at the initial posttreatment acquisition had persistent cancer at 6-month biopsy.

Conclusion: MR imaging results of the prostate show a consistent sequence of changes after treatment with HIFU and can provide information to the operator about completeness of treatment.

There is currently little to offer men with localized prostate cancer between the two extremes of watchful waiting and radical treatment—most commonly prostatectomy or radiation therapy (1). Ablation of the gland has been proposed as an alternative that has the potential to completely treat the tumor while minimizing the sexual and urinary morbidity that still accompany established radical therapies (2). Several techniques have been used in the prostate—including microwave (3) and radiofrequency (4) ablation, cryotherapy (5), photodynamic therapy (6), and high-intensity focused ultrasound (HIFU) treatment (7).

HIFU is, in several respects, ideally suited to the prostate. In contrast to extracorporeal devices for the liver and kidney (8), with the transrectal approach, there is little movement of the target because of respiration or reflection by overlying bone. A focal distance of 3 or 4 cm allows the generation of coagulative necrosis in treatment voxels less than 0.2 mL and allows a treatment volume that conforms to the shape of the prostate (9)—a degree of precision that may be beyond that of other techniques. Even so, complete ablation is likely to affect periprostatic tissues, including the neurovascular bundles containing the cavernosal nerves (10) and the external urethral sphincter. Preservation of these structures—and the patient’s erectile and urinary function—must be balanced against full treatment of the gland.

Although impotence rates after HIFU treatment approach 50% (11), it is likely that in its current clinical implementation, the prostate is not being fully ablated: In published series, the recurrence rates for cancer range between 25% and 38% (7,11,12). To our knowledge, no groups have reported mean reductions in prostate volume of more than 50% (12,13), and several groups have found it difficult to treat the anterior gland (14).

If we are to improve outcomes, a fundamental requirement for HIFU treatment (and ablative technologies in general) is a method that provides anatomic information to the operator about areas that have been over- or undertreated. This might lead to modifications in future technique, and if obtained soon after treatment, might indicate the need for further ablation. Such a method might also help predict outcome earlier than established measures, such as prostate-specific antigen (PSA) measurement and biopsy.

Magnetic resonance (MR) imaging has great potential in this setting, and Rouviere et al (14) have described the appearance of the prostate on contrast material–enhanced MR images obtained up to 5 months after HIFU treatment. Rouviere et al found a good correlation between the theoretical treatment volume and the volume of nonenhancing prostate on a subsequent acquisition. The aim of our study was to prospectively evaluate MR imaging findings after HIFU treatment of the prostate and to correlate them with clinical and histologic findings.

 

MATERIALS AND METHODS

Misonix (the European distributors of the Sonablate device) funded the phase-II European study and provided equipment and reimbursed the hospital for costs. The company has funded two authors (I.M.H. and R.O.I.) through educational awards. One author (M.E.) has acted as a paid consultant to Misonix and also received honoraria for training and teaching. Authors other than I.M.H., R.O.I., and M.E. had control of the information and data submitted for publication. Misonix was not involved in the analysis of data or the writing of this article.

Patients

We included the first 15 men at University College Hospital (age range, 46–70 years; mean age, 59 years) who were taking part in a registered phase-II multicenter European study of HIFU therapy for organ-confined prostate cancer (Table 1). The study was approved by the local ethics committee, and full written consent was obtained from each patient. The patients understood that HIFU is an experimental treatment whose long-term outcome is unknown and were offered full conventional treatment as an alternative. The study was limited to men with a serum PSA level 15 μg/L or less, Gleason score less than 8, prostate volume less than 40 mL, life expectancy more than 5 years, and age less than 80 years. There was no limit to the number of biopsy cores that had a positive finding or the amount of cancer in each core removed. Patients with a history of previous prostate surgery were excluded, as were men who had undergone androgen deprivation therapy in the 6 months prior to recruitment or had intragland prostatic calcification more than 1 cm in diameter.

Table 1.  Patients and Demographics

 table 1

 * Ratio of cores with a positive finding to cores obtained.

 † Image not available for analysis; volume was calculated by using US measurements.

The Sonablate 500 (Focus Surgery, Indianapolis, Ind) consists of a power generator, water cooling system (the Sonachill), a treatment probe, and a positioning system. The probe contains two curved rectangular piezoceramic transducers with a driving frequency of 4 MHz and focal lengths of 30 and 40 mm. During treatment, these may be driven at low energy to provide real-time diagnostic imaging or at high energy for therapeutic ablation (in situ intensity, 1300–2200 W/cm2). The probe is covered with a condom, under which cold (17°–18°C) degassed water is circulated to help protect the rectum from thermal injury.

Patients were prepared before the procedure with two phosphate enemas to empty the rectum. Oral bowel preparation was used in some patients. Treatment was performed with general anesthesia in the lithotomy position and was performed or closely supervised in every case by an author (M.E., 2 years of experience in HIFU treatment). After gentle dilation of the anal sphincter, the treatment probe was introduced with a covering of ultrasonographic (US) gel to couple it to the rectal mucosa and was held in position with an articulated arm attached to the operating table. A 16-F Foley urethral catheter was inserted using sterile technique, and a 10-mL balloon was inflated to allow the bladder neck and median sagittal plane to be seen accurately. It was removed before treatment began.

Treatment was planned by using US-acquired volumes consisting of stacks of both sagittal and transverse sections (voxel size, 2 × 3 × 30 mm) and was applied in rows that extended in the craniocaudal axis, interleaved to avoid interference from adjacent, recently treated areas. After each 3-second period of ablation, diagnostic transverse and sagittal images in the plane of treatment were obtained to permit tailoring of the energy delivery in the next voxel according to visible changes on the gray-scale image. This is an important difference from the device used by Rouviere’s group (14), in which power is planned before the treatment begins. We aimed to set the power for each voxel at a level that produced hyperechoic change due to cavitation (as described by Illing et al [15]), and we invariably treated the whole anterior prostate. Neurovascular bundles were not identified at treatment (the Sonablate device does not yet have color Doppler capability); rather, we aimed to avoid treating outside the capsule where they lie posterolaterally (10). The time between the first ablation and the point at which treatment was considered complete was 3.0–4.4 hours (mean, 3.6 hours). A 16-F urethral catheter was placed immediately after the treatment and was left in place for 2 weeks.

MR Imaging

For most preoperative examinations and for all post-HIFU imaging, we used an MR machine (Symphony or Avanto; Siemens, Erlangen, Germany) with 1.5-T magnet and a pelvic-phased array coil. Except where stated, a full protocol of T1- and T2-weighted turbo spin-echo (Siemens) images and a dynamic fat-saturated postcontrast volume acquisition were used for both preoperative diagnostic and planning imaging and for postoperative assessment of HIFU treatment (Table 2). The contrast material used was 20 mL of gadopentetate dimeglumine (Magnevist; Schering, Berlin, Germany) given intravenously at 3 mL/sec.

Table 2. MR Sequences Used at Prostate Imaging

table 2

We aimed to image patients less than 1 month after treatment and did so in 12 patients. The remaining three patients were imaged between 1 and 3 months after treatment. Two patients were imaged in both time periods. Every patient underwent a 6-month MR examination.

Image Analysis

All volume measurements (except where stated) were acquired by using planimetry of contiguous 3-mm sections (16). T2-weighted images were used for measurement of prostatic volume both before and after treatment. The amount of intermediate- or high-signal-intensity material (ie, higher than muscle) remaining within the prostate was also measured on the 6-month posttreatment T2-weighted image.

The volume of nonenhancing prostate tissue at the post-HIFU acquisition was measured by using the final dynamic postcontrast image. On the initial posttreatment image, we also measured the volume of extraprostatic tissue that was both of low signal intensity on the T1-weighted image and nonenhancing. The distance between this tissue and the rectal mucosa was measured at its narrowest point. The mean thickness of the enhancing rim surrounding the treatment volume was measured on transverse postcontrast T1-weighted spin-echo images and was calculated by dividing the area of the rim by its circumference.

The volume of persistently enhancing prostate tissue on the initial image was calculated by subtracting the nonenhancing volume from the total volume of prostate on the T2-weighted image. This could be calculated in 13 patients; one patient did not receive contrast material at the post-HIFU MR acquisition, and the other was imaged more than 2 months after treatment.

All measurements were performed by a first-year radiology fellow (A.P.S.K.) without knowledge of PSA and histologic results. Two other observers independently measured the three key parameters that were used for correlation calculations for each patient: (a) the volume of nonenhancing prostate on the initial image, (b) the total volume of the prostate on the initial image, and (c) the final prostate volume at 6 months. One was a consultant uroradiologist with more than 10 years of experience in the interpretation of prostate MR images (C.A.); the other was a third-year urology research fellow with an interest in prostate imaging (R.O.I.). For each parameter, the mean of the three observers’ measurements was calculated and used for further analysis.

PSA Measurement and Prostate Biopsy

Serum PSA level was measured before and at 1.5, 3, and 6 months after HIFU treatment. The nadir was defined as the lowest of the three values.

Biopsies were performed by an author (A.P.S.K., with 4 years of experience in prostate biopsy) by using a transrectal approach with US guidance and an 18-gauge needle with a 2-cm throw soon after the 6-month MR examination. The number of cores obtained depended on the amount of residual prostate and varied between two and 10 (median, eight cores).

Erectile Function and Continence

The International Index of Erectile Function was used to assess erectile function both before and 3 months after HIFU treatment in each patient (17). The most important question was, “How often were your erections hard enough for penetration [with or without phosphodiesterase type 5 inhibitors]?” A score of 2 (a few times in 4 weeks) to 5 (always) was, for the purposes of this article, considered evidence of intact erectile function.

Men were asked to complete the International Continence Society–validated continence function questionnaire at baseline and at 3 and 6 months after therapy. The question deemed to be most informative was how often the patient required the use of pads or adult diapers. Responses could include “never,” “not more than one per day,” “1–2 per day,” or “more than 3 per day.”

Statistical Analysis

To assess the variance of results between observers, we used the intraclass correlation coefficient (18) applied to measurements obtained by three observers of the calculated volume of enhancing prostate on the initial post-HIFU image and the 6-month prostate volume.

The Spearman rank test was used to assess the correlation between enhancing prostate volume and serum PSA level nadir, and the Pearson test was used to examine the correlation between initial enhancing prostate volume and final prostate volume. Only the patients who were imaged less than 1 month after treatment were included in the analysis. These tests were performed by using software (GraphPad Prism for Mac, version 3; http://www.graphpad.com).

Because some of the covariance of volumes measured after treatment was likely to be due to their correlation with pretreatment prostate volume, we also applied a correction: The values were expressed as a proportion of the pretreatment volume, and a further correlation measurement was performed by using the Pearson test. In each case, a P value of less than .02 was considered to indicate a significant difference.

 

RESULTS

Up to 1 Month After Treatment

T2-weighted images.—Compared with that on the preoperative image, the prostate volume increased in every case (Table 1 and Table E1, Fig 1). The signal intensity from the prostate on T2-weighted images within the first month was always heterogeneous and variable. It was impossible to predict from the findings on T2-weighted images which areas of the prostate would enhance after intravenous contrast material administration. The periprostatic fat was also heterogeneous in signal intensity, which was consistent with edema (Fig 2).

Figure 1: Graph of change in prostate volume after HIFU treatment. Volume rises initially (less than 1 month after treatment) and is reduced in all cases at 6 months. Numbers = patient numbers.

 Picture1

Figure 2: MR images in patient 1 (a–d) and (e–h) patient 8 show low volume of enhancing prostate at initial imaging and small residual prostate at 6 months. Posttreatment serum PSA level was less than 0.05 μg/L in both cases.

Figure 2a:

Picture2a

Figure 2b:

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Figure 2c:

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Figure 2d:

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Figure 2e:

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Figure 2f:

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Figure 2g:

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Figure 2h:

Picture2h

T1-weighted images.—The prostate was of predominantly low signal intensity, although patchy areas of intermediate or high signal intensity, likely to represent hemorrhage, were a constant finding within the gland and in all but one of 28 seminal vesicles.

Postcontrast images.—In each patient, the postcontrast images showed a central area of nonenhancing tissue. This conformed to the treatment volume and was surrounded by an enhancing rim of mean thickness of 2–8 mm (median, 4 mm) that was continuous around the prostate in most patients (Fig 2; Table E1,).

The enhancing prostate varied in size and position. Part of the enhancing rim usually lay within the prostatic capsule and continued to the prostatic apex where there was almost always some enhancing tissue between the nonenhancing prostate and the external urethral sphincter. In many patients, more central areas of enhancement were seen: at the apex or base, either posteriorly or anteriorly (Table E1), and were almost always in continuity with the rim.

In every patient, the nonenhancing, low signal intensity within the prostate extended outside the gland and involved the periprostatic fat and the levator ani muscle, particularly anterolaterally (Table E1, Figs 23). This varied considerably and tended to be most prominent in those who had no residual gland enhancement and had an undetectable serum PSA level after HIFU treatment (Table E1). In several patients, the nonenhancing area extended to involve the Denonvilliers fascia. (The distance between its margin and the rectal muscle is listed in Table E1.) In one patient, a proportion of the rectal wall enhanced avidly, but in no patient was there loss of rectal wall enhancement to suggest necrosis.

Figure 3: MR images obtained near the prostate apex show incomplete treatment and persisting high signal intensity in prostate. Serum PSA level nadir = 0.61 μg/L.

Figure 3a: Patient 4:

 Picture3a

 Figure 3b: Patient 4:

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Figure 3c: Patient 4:

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Figure 3d: Patient 4:

Picture3d

At 1–3 Months

In three patients, there was a “double rim” (Fig 4) on postcontrast images obtained at 36 and 56 days after HIFU treatment. The inner component lay within the prostate and the outer at the prostatic capsule; the intervening part was of low signal intensity on both T1- and T2-weighted images.

 Figure 4: MR images of “double rim” at 56 days after HIFU treatment.

Figure 4a: Patient 3:

 Picture4a

Figure 4b: Patient 3:

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Figure 4c: Patient 3:

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Six-month Appearance

T2-weighted images.—In every patient, the volume of the prostate was reduced by more than 45% (median, 61% reduction) (Table E1). On T2-weighted images, the majority of the persisting prostate was of low signal intensity, with poor definition to the capsule and with persisting heterogeneous signal intensity to the surrounding fat. However, in 12 of 15 patients, there was persisting high or intermediate signal intensity of the prostate—up to 5.34 mL in volume and most often seen posteriorly and at the apex (Table E1, Figs 3 and 5). In many patients (for example, those in Fig 2), low-signal-intensity prostate of reduced volume surrounded a capacious prostatic cavity continuous with the urethra, which is similar to the cavity seen after transurethral resection (19).

Figure 5: MR images of incomplete treatment of tumor and positive biopsy findings in three of 10 cores at 6 months (in right lateral midzone, right lateral base, and right parasagittal base samples). Serum PSA level nadir = 1.19 μg/L.

Figure 5a: Patient 13:

Picture5a

Figure 5b: Patient 13:

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Figure 5c: Patient 13:

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Figure 5d: Patient 13:

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Figure 5e: Patient 13:

Picture5e

Postcontrast images.—Some small areas of nonenhancing tissue persisted in eight of 14 patients, but this was less than 1 mL in all but one (patient 13, in whom 4 mL of the gland volume of 18.7 mL was nonenhancing). The levator muscle showed a normal signal intensity.

Correlation Between Initial Imaging and Later Findings

In the 12 patients who underwent the initial acquisition within 1 month of HIFU treatment, the volume of enhancing tissue on the initial posttreatment image was positively correlated with the serum PSA level nadir (Fig 6) (Spearman r = 0.90, P < .001) and with the amount of residual tissue at 6 months (including all low-signal-intensity material that was likely to represent fibrosis or necrosis) (Fig 7) (Pearson r = 0.80, P = .001).

 Figure 6: Graph of relationship between the proportion of the prostate still enhancing on initial image and serum PSA level nadir. There is a significant positive correlation (Spearman r = 0.90, P < .001). * = patient 13, who was included in graph but not in analysis (imaged 56 days after HIFU treatment). Patients 14 and 15 are not included because they did not undergo contrast-enhanced acquisition within 2 months of HIFU treatment. μgl−1 = μg/L.

 Picture6

Figure 7: Graph of relationship between the proportion of the prostate still enhancing on initial image and final volume of prostate. There is a significant positive correlation between the variables (Pearson r = 0.80, P = .001). * = patient 13, who was included in graph but not in analysis (imaged 56 days after HIFU treatment). Patients 14 and 15 are not included because they did not undergo contrast-enhanced acquisition within 2 months of HIFU treatment.

Picture7

When posttreatment volumes are expressed as a proportion of pretreatment prostate volume, the correlation between enhancing tissue volume on the initial posttreatment image and the 6-month prostate volume persists (Pearson r = 0.70, P = .001).

Interobserver Correlation

The interobserver variation was excellent for the calculated volume of prostate enhancing on the initial post-HIFU image, with an intraclass correlation coefficient of 0.92, and was good for final prostate volume (intraclass correlation coefficient = 0.73).

Clinical Findings

In five patients (patients 1, 3, 8, 11, and 13), there was imaging evidence (at MR imaging or retrograde urethrography) of a stricture in the mid- or distal prostatic urethra, which was confirmed by using flow rate studies and treated by using self-catheterization or with graded urethral dilators. None have required formal urethrotomy. Patient 14 developed a bladder neck stricture, which was treated successfully by incision.

Before treatment, no men required pads or adult diapers for incontinence. At 6 months after the treatment, four men still required not more than one pad per day. In two cases, this was for reassurance rather than actual leakage.

In the 14 patients in whom there was intact erectile function (score 2–5 for the question, “How often were your erections hard enough for penetration?”) before HIFU treatment, it was intact in nine patients after the procedure. One patient had stopped trying to achieve erections, and four could not achieve penetration.

Histologic Findings

In the three patients in whom there was no high-signal-intensity peripheral zone at 6 months and with serum PSA level less than 0.05 μg/L, there was either no prostatic tissue or only a small group of acini in one core. The remaining patients had a variable amount of residual prostate at core biopsy.

Five patients had residual tumor. In three patients, it was seen in at least two cores (Table E1), and these three patients also had the largest volume of enhancing prostate on the initial post-HIFU MR image (Figs 6 and 7) and more than 2 mL of intermediate- or high-signal-intensity gland on T2-weighted images at 6 months.

In four of five patients with residual cancer, it could not be identified on either contrast-enhanced or T2-weighted images. In one patient (Fig 4), the early dynamic images showed prominent enhancement in the anterior gland, which was consistent with residual cancer found at the distal (ie, nonrectal) end of three right-sided biopsy cores. Such enhancement was not seen in patients with no cancer found at core biopsy.

 

DISCUSSION

We found a consistent sequence of changes at MR imaging after HIFU treatment of the whole prostate. The proportion of enhancing tissue on the initial posttreatment MR image was predictive of gland volume at 6 months and serum PSA level nadir. A strong statistical relationship between the latter and outcome has recently been demonstrated (20).

Most patients with residual cancer had evidence of incomplete ablation early (a large volume of enhancing prostate on the initial image) and late (a large volume of high-signal-intensity residual prostate on T2-weighted images at 6 months).

In some patients it was possible to achieve an undetectable serum PSA level at 6 months and entirely low signal intensity on T2-weighted images in the region of the prostate. These patients had either no or a small amount of viable prostate in one core at biopsy.

Conversely, in spite of reductions in prostate volume of more than 45% at 6 months, the majority of patients had histologic evidence of persisting viable prostate, and in a group of patients with organ-confined disease but no limit to the volume of cancer pretreatment, one-third had evidence of residual tumor.

Persisting enhancing prostatic tissue usually occurred at the periphery (or extended toward the center of the gland from it) and was particularly common at the apex and near the rectum.

Results of one previously published study (14) of post-HIFU appearances with MR imaging show a similar sequence of acute changes, although there was no attempt to quantify prostate volume at 6 months. There is also a large body of work on the MR imaging appearances with thermotherapy (whether laser [21,22] or radiofrequency [23]) and cryotherapy (24) within the prostate and other organs. The hyperenhancing rim of tissue is a constant finding in several tissues, including the liver (25), the kidney (26), and the brain (27). In the liver and the kidney, it is thin (1 mm or less) and, in most cases, has disappeared by 2 months after ablation (28). Within the prostate, the hyperenhancing rim has been shown to occur after laser ablation of benign prostatic hyperplasia (21,22) and after HIFU treatment (14).

Histologic evidence in animal models—including rabbit and porcine liver (29)—suggests that the enhancing rim corresponds to an area of inflammation and then fibrosis, with a variable amount of residual, viable tissue. How much of the rim will be viable after ablation of the prostate in humans remains uncertain. On the one hand, after HIFU treatment, core biopsy results show “partial or complete necrosis” in the rim (14). On the other, after laser ablation of benign prostatic hyperplasia, the volume of coagulative necrosis at histologic examination correlates very well with the central nonenhancing region at MR imaging, not including the rim (22). The answer is likely to be that a variable amount of the rim contains viable tissue (depending on the organ being imaged [30], the nature of the treatment, and the interval before the acquisition), and the implication is that the only reliably necrotic area at MR imaging is that which does not enhance. We have avoided the term necrosis for the nonenhancing areas of prostate seen in our current study, but from these data it is likely that the areas of prostate without enhancement are truly necrotic.

The distribution of enhancing prostate on posttreatment MR images fits with histologic evidence that “ventral, lateral and dorsal sides of the prostate” have residual viable prostatic tissue at histologic examination after HIFU treatment (31). What all of these areas have in common is proximity to the more richly vascular prostatic capsule. Is it possible that increased vascularity here results in reduced efficacy? This is another area that has been addressed by Rouviere’s group (32), who did not find a correlation between successful ablation and prostate vascularity by using power Doppler US; they conclude, as others have (33,34), that short (3-second) high-intensity bursts of focused ultrasound are unlikely to be markedly affected by blood flow. An alternate explanation is a geometric one: Centrally lying voxels are easier to treat because they may be rendered necrotic either by direct treatment or by damage to supplying vessels in the periphery.

An implication of these results is that the best strategies for minimizing complications while ensuring destruction of the cancer are likely to involve a degree of targeting: If the tumor can be imaged with MR imaging, the patient might be treated with higher power and wider margins (including periprostatic fat, muscle, or even neurovascular bundles) at the site of the cancer and with a standard intensity to the rest of the gland. An analogous approach is the wide excision, including a unilateral neurovascular bundle, of bulky tumors at radical prostatectomy (35). Such an approach may well have benefited our patients 7 and 13.

One methodologic issue that is currently unresolved relates to the timing of MR imaging. A detailed within-patient study of MR imaging changes after HIFU treatment is needed to properly describe the longitudinal changes in the appearance of the prostate. Rouviere et al (14) found that the area of nonenhancing tissue decreases by 50% at 1 month compared with that at an immediate (<1 week) post-HIFU acquisition, which suggests that for an accurate assessment of necrosis volume, the prostate should be imaged as soon as possible after treatment. Of course, perfusion would ideally be assessed during HIFU treatment so that undertreated areas could be further ablated. There is some evidence that Doppler or contrast-enhanced US (36) could play this role, but, to our knowledge, there are no studies on the correlation of immediate findings with later clinical data, such as serum PSA level or histologic examination.

We used fast low-angle shot sequences to assess enhancement because we found that the subjective assessment (together with objective measurements of signal intensity) of the dynamic series helped us identify truly nonenhancing tissue. However, the T1-weighted spin-echo postcontrast sequence would have been adequate, and we consider, as others do (22), dynamic contrast-enhanced sequences not to be an essential part of the protocol for postablation assessment. What is certain is that unenhanced T2-weighted sequences are inadequate for assessing necrosis (14,22).

Our results differ from those of other published series of HIFU treatment in the marked reduction in gland volume and absence of zonal anatomy in many patients observed at 6 months. In contrast to the study of post-HIFU MR imaging by Rouviere et al (14) who used a different device, we did not find that “HIFU-induced abnormalities seem to disappear within 3–5 months.” Rather, in several patients, it was difficult to discern any residual prostate at all at both MR and US studies. The difference probably lies in the power used for treatment and the completeness of gland coverage. The stricture rate of six of 15 is high when compared with that in published series (7,37,38) and may be related to the power used, the degree of fibrosis occurring in the prostate, and the strategy for catheterization. The latter is considered likely to be important, and we have recently changed to using a suprapubic catheter (rather than urethral) after treatment. The rate of impotence after treatment is similar to that in published series (11), as is grade I incontinence.

Our work has implications for the conduct of HIFU. The finding that the volume of enhancing prostate on the initial posttreatment image correlates well with intermediate measures, such as serum PSA level nadir and biopsy evidence of residual cancer, suggests that MR imaging can provide the operator with feedback on the effectiveness of the intervention. This information might enable modification of the technique to treat areas that have been incompletely ablated in previous patients—in our series, those areas encompassed the apex and posterior gland and rarely anterior tissue (in contrast to other study results [14]). Conversely, we might have reduced power or treatment volume at the anterolateral aspect of the gland adjacent to the levator muscle. Such feedback has been cited as a desirable attribute for ablation technology (39) and up to now has been missing.

Our study had several limitations. Although it is likely that nonenhancing areas at MR imaging represent necrosis, we do not have direct histologic evidence. Sampling error and misregistration limit the utility of core biopsies in this context. We have shown that the MR imaging appearances soon after HIFU treatment correlate with findings at 6 months, but this is not the same as outcome. A considerably longer follow-up and a larger number of patients will be necessary to determine both the ultimate efficacy of HIFU treatment and the ability of MR imaging to help predict outcome. Last, while our findings suggest that MR imaging soon after treatment may be useful to assess areas of under- and overtreatment, this is not real-time feedback and does not allow modification of the treatment as it progresses.

In summary, MR imaging results in the first 6 months after HIFU treatment show a consistent sequence of changes, and appearances in the 1st month correlate with serum PSA level nadir and imaging findings at 6 months. Such imaging results hold promise for providing feedback to the operator about the effectiveness of treatment.

 

ADVANCES IN KNOWLEDGE

  • Treatment of prostate cancer by using ablation with high-intensity focused ultrasound (HIFU) results in a consistent series of changes within the gland during 6 months seen at contrast-enhanced MR imaging.
  • Within 1 month after treatment, a central nonenhancing area is surrounded by an enhancing rim of tissue lying variably within and outside the prostate.
  • At 6 months, the gland is markedly smaller and of partly or completely low signal intensity on T2-weighted images.
  • The amount of enhancing prostate on the initial image correlates with several findings at 6 months, including serum prostate-specific antigen level nadir and prostate volume.

 

IMPLICATION FOR PATIENT CARE

  • MR imaging after HIFU treatment may provide information about completeness of tumor ablation and the need for early retreatment or close monitoring in cases of incomplete coverage.

 

Footnotes

  • Trial registration: This trial started recruiting before the trial registration requirements of the International Committee of Medical Journal Editors were formalized.

See Materials and Methods for pertinent disclosures.

Author contributions: Guarantors of integrity of entire study, A.P.S.K., I.M.H., C.A.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, A.P.S.K., M.E., I.M.H., R.O.I., C.A.; clinical studies, A.P.S.K., R.O.I., C.A.; statistical analysis, A.P.S.K.; and manuscript editing, all authors

Abbreviations:HIFU = high-intensity focused ultrasoundPSA = prostate-specific antigen

 

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