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Research Corner Abstracts by Bruce Shriver and Tom Swartz
In this issue:
The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas
The investigators of this study
analyzed the PI3K-AKT signaling cascade in a cohort of sarcomas. They found
a marked induction of insulin receptor substrate-2 (IRS2) and phosphorylated
AKT and a concomitant upregulation of downstream effectors in most
leiomyosarcomas. To determine the role of aberrant PI3K-AKT signaling in
leiomyosarcoma pathogenesis, they genetically inactivated
Pten in the smooth muscle cell
lineage by cross-breeding PtenloxP/loxP
mice with Tagln-cre mice.
Mice carrying homozygous deletion of Pten
alleles developed widespread smooth muscle cell hyperplasia and abdominal
leiomyosarcomas, with a very rapid onset and elevated incidence (
Rhabdomyosarcoma Cancer Stem Cell Identified As published in the June 1 issue of Genes & Development, Dr. Leonard Zon (Children's Hospital Boston) and colleagues have identified the cancer stem cell for rhabdomyosarcoma, the most common soft-tissue sarcoma of childhood. "Identifying the cancer stem cell and the evolutionarily conserved genetic programs underlying self-renewal in ERMS will likely lead to new insights into how to destroy these cell types in established malignancies," explains Dr. Zon. Rhabdomyosarcoma (RMS) is an aggressive cancer that arises from a primitive skeletal muscle cell called a "rhabdomyoblast". Depending upon on the histology of the cancerous cells, there are several different subtypes of RMS. Embryonal rhabdomyosarcoma (ERMS) is the most common subtype, usually found in children under 15, in the head and neck region and genitourinary tract. Dr. Zon and colleagues have developed an animal model to identify and test therapeutic targets of human ERMS. The scientists artificially activated the RAS pathways to induce ERMS in a strain of genetically engineered zebrafish. Some transgenic zebrafish developed visible tumors by 10 days of age. Through their model, Dr. Zon and colleagues were able to identify both an ERMS tumor-cell-of-origin and a novel genetic signature that underlies ERMS progression in zebrafish and human patients. Cancer stem cells make up only a small fraction of the overall number of cells in a tumor. However, they are capable of giving rise to other cancer cells, and thereby drive tumor growth and metastasis. To prevent recurrence and progression, effective long-term therapies must target the self-renewing population of cancer stem cells. The zebrafish is ideally suited for use in targeted chemical genetic approaches to specifically inactivate cancer pathways we have identified in our study. Identifying drugs that inactivate these pathways in the ERMS cancer stem cell may have far reaching implications for treatment of patients with this disease."
Stem cell-associated genes are extremely poor prognostic factors for soft-tissue sarcoma patients Cancer stem cells can play an important role in tumorigenesis and tumor progression. However, it is still difficult to detect and isolate cancer stem cells. An alternative approach is to analyze stem cell-associated gene expression. The investigators of this study investigated the coexpression of three stem cell-associated genes, Hiwi, hTERT and survivin, by quantitative real-time–PCR in 104 primary soft-tissue sarcomas (STS). Multivariate Cox's proportional hazards regression analyses allowed correlating gene expression with overall survival for STS patients. They found that coexpression of all three stem cell-associated genes resulted in a significantly increased risk of tumor-related death. Importantly, tumors of patients with the poorest prognosis were of all four tumor stages, suggesting that their risk is based upon coexpression of stem cell-associated genes rather than on tumor stage.
Should Soft Tissue Sarcomas Be Treated at High-volume Centers?: An Analysis of 4205 Patients Using a large prospective cancer registry, the effects of surgical volume on patient outcomes for soft tissue sarcoma (STS) were examined in this study. A significantly increased overall short-term and long-term survivals as well as improved limb preservation were observed at high-volume centers. This study suggests that soft tissue sarcoma should be treated at high-volume centers. STS cases registered in the Florida Cancer Data System (FCDS) between 1981 and 2001 were analyzed. Medical facilities were ranked by STS operative volume. Facilities above the 67th percentile for volume were defined as high-volume centers (HVCs). The Results were as follows: Of the 4205 operative cases of STS identified, 68.1% were treated at low-volume centers (LVCs) and 31.9% at HVCs. A larger proportion of high-grade tumors (53.8% vs. 44.3%) and lesions over 10 cm (40.7% vs. 28.7%) were resected at HVC (P < 0.001). The 30-day mortality was 0.7% for HVC and 1.5% for LVC (P = 0.028), and mortality rates at 90 days were 1.6% and 3.6%, respectively (P = 0.001). Median survival was 40 months at HVC and 37 months at LVC (P = 0.002). Univariate analysis demonstrated significantly improved survival at HVC for high-grade tumors (median 30 months vs. 24 months, P = 0.001), lesions over 10 cm (28 months vs. 19 months, P = 0.001) and truncal or retroperitoneal sarcomas (39 months vs. 31 months, P = 0.011). Limb amputation rate was lower (9.4% vs. 13.8%, P = 0.048) and radiation and chemotherapy were more frequently administered at HVC (OR = 1.54). On multivariate analysis, treatment at a HVC was a significant independent predictor of improved survival (OR = 1.292, P = 0.047). The authors Conclude: STS patients treated at HVC have significantly better survival and functional outcomes. Patients with either large (>10 cm), high-grade or truncal/retroperitoneal tumors should be treated exclusively at a high-volume center.
National Research Study On Causes Of Osteosarcoma Cancer In Children Led By University of Minnesota Logan Spector, Ph.D., a University of Minnesota Cancer Center researcher, has received a $1.7 million grant from the National Cancer Institute (NCI) to lead the largest and most comprehensive study to date on the causes of pediatric osteosarcoma. Spector is an assistant professor of pediatrics at the University of Minnesota Medical School and Cancer Center. The University of Minnesota is a national research and treatment site for children with bone cancer. This four-year research study will include 500 children in the United States and Canada who have been diagnosed with osteosarcoma. Their parents also will be enrolled in the study. "Previous research has shown a close correlation between the childhood growth curve and osteosarcoma," Spector says. "Children with a more rapid or sustained growth spurt may have a higher risk of osteosarcoma. Our research will investigate the effects of genes related to bone growth to determine if they have a role in causing this cancer." Spector says. "We think there could be a link between bone growth genes and osteosarcoma because the timing and extent of adolescent bone growth are mostly genetic. “We also will study how well the cells repair DNA damage naturally, and if the genes are impacted by diet, physical activity, family health history, and other lifestyle-related habits." A DNA sample will be obtained from each child with osteosarcoma as well as from the parents to learn if the children inherited these bone growth genes more or less often than expected. Growth records of children with the disease will also be compared to national standards, and all participants will complete a questionnaire about their family history and lifestyle. Spector will work with other pediatric cancer researchers at the University of Minnesota; The Hospital for Sick Children in Toronto, Canada; Cincinnati Children's Hospital Medical Center in Cincinnati, Ohio; Memorial Sloan-Kettering Cancer Center in New York; and the NCI. The Twin Cities-based Children's Cancer Research Fund and the Karen Wyckoff Rein in Sarcoma Fund provided funding for the preliminary study.
Scientists Directly Target Cancer Cells Using Nanotechnology An Australian biotechnology firm announced on May 10th that it had developed a means of delivering anti-cancer drugs directly to cancer cells, which aims to avoid the debilitating toxicity associated with chemotherapy. The method uses nanotechnology, which involves molecules far smaller than a human cell. Direct targeting of chemotherapy drugs would allow dosages thousands of times lower than that in conventional chemotherapy and be more easily tolerated by patients. Writing in the May issue of Cancer Cell journal, the biotech firm EnGeneIC said it had developed nano-cells containing chemotherapy drugs. Via antibodies on their surface, these nano-cells target and latch on to cancer cells. Once attached, the nano-cell is engulfed and the drug is released directly inside the cancer cell. The firm said the bacterially derived nano-cell, called EnGeneIC delivery vehicles, had proven safe in primate trials and resulted in significant cancer regression. It hopes to carry out human trials later in 2007 if it gains approval from Australian, U.S., European and Japanese regulatory authorities. "For the first time there is a real possibility that this technology could lead to the use of multi-drug combinations and eventual custom-made therapies in cancer patients," research scientist Jennifer MacDiarmid said in a statement. MacDiarmid also expressed hope that the technology could be used to attack the problem of drug resistance exhibited by late stage cancers.
Does Length of Symptoms Before Diagnosis of Sarcoma Affect Patient Survival? It generally is accepted that earlier diagnosis of sarcoma is associated with a better disease-free survival. Because the legal community is convinced this is true, a delay in cancer diagnosis is a frequent source of medical malpractice lawsuits. The investigators of this study asked whether symptom duration before diagnosis affected sarcoma outcome. They compiled prospective data from 624 consecutive patients with sarcoma treated from 1992 to 2003. The data included length of symptoms before diagnosis, tumor size and grade, presence of metastatic disease at diagnosis, anatomic location, disease-free survival, overall survival, and local recurrence. They investigators found that length of symptoms did not predict presence of metastatic disease at diagnosis, survival, or disease-free survival. They found no correlation between the length of a patient's symptoms and the tumor size or anatomic site of disease (axial, proximal, and distal). Tumors were larger at diagnosis in axial and proximal locations than in more peripheral locations, despite no difference in length of symptoms before diagnosis. Patients with sarcoma with long-standing symptoms did not have a worse prognosis than those with a shorter length of symptoms before diagnosis in terms of disease-free survival, overall survival, and presence of metastatic disease at diagnosis.
Low Levels of Common Enzyme Key to Resistance in Ewing's Sarcoma A study from scientists at the University of Freiburg, Germany, and their collaborators at the National Cancer Institute has pinpointed a potential mechanism for resistance of Ewing’s sarcoma to a protein that may be useful in fighting cancer -- and a possible method for overcoming this resistance. The results appear in the June 2007 issue of the American Journal of Pathology.
The protein, called TRAIL (tumor necrosis factor apoptosis-inducing ligand), has generated tremendous interest among scientists looking for new therapies that target cancer cells but spare normal dividing cells. TRAIL binds to receptors on the surface of cancer cells and sets off a series of signals that cause the cells to commit suicide. Almost all normal cells are unaffected by TRAIL. The cells of many solid tumors, including Ewing’s sarcoma, have shown extreme sensitivity to TRAIL in laboratory experiments, and investigators have started testing the protein in early clinical trials.
The scientists who conducted the study, led by Udo Kontny, M.D., from the University of Freiburg, showed that Ewing’s sarcoma cells that express low levels of an enzyme called caspase-8, which plays a role in cell suicide (apoptosis), are resistant to TRAIL-induced killing. However, the addition of interferon-gamma, a protein produced by cells of the immune system, to treatment with TRAIL caused the resistant cells to produce more caspase-8, making them once again sensitive to TRAIL-induced death.
“This study is an excellent example of how modern molecular biology can help us unlock the detailed mechanisms driving the new, targeted therapies for cancer,” said NCI Director John E. Niederhuber, M.D. “If a targeted treatment that shows promise in the laboratory does not work as expected in clinical trials, we need to go back and understand what properties of the cancer cells might be driving resistance.” “When you look in the test tube, it’s really quite remarkable how quickly and dramatically Ewing’s sarcoma is killed by TRAIL,” explained Crystal Mackall, M.D., from NCI’s Center for Cancer Research (CCR), whose laboratory headed the NCI portion of the investigation. “But the initial results in animal studies were just not as effective as we would have expected. So we ended up thinking that, however this tumor is becoming resistant, it must be really important, and we’re going to need to add something to improve the effectiveness of TRAIL treatment in the clinic.”
The scientists focused on caspase-8 because lack of the protein had been linked in laboratory studies to TRAIL resistance in Ewing’s sarcoma cells and the cells of other tumors. To see if caspase-8 expression is also limited in tumor tissues acquired from patients, the researchers measured the expression of caspase-8 in 54 tissue samples taken from 47 patients with Ewing’s sarcoma. They found that, while 50 out of the 54 samples expressed caspase-8, the number of cells within each sample that expressed the protein varied considerably. In 76 percent of the samples, caspase-8 expression was detected in 60 to 100 percent of the cells. In the other 24 percent of the samples, caspase-8 expression was detected in only 0 to 50 percent of the cells. Therefore, within any individual Ewing’s sarcoma tumor, cells that lack caspase-8 could cause resistance to TRAIL.
The investigators next tested whether interferon-gamma, which has been shown in the laboratory to increase caspase-8 expression in cells, could sensitize Ewing’s sarcoma cells with low expression of caspase-8 to treatment with TRAIL. They found that doses of interferon-gamma, within the range easily tolerated by patients, increased caspase-8 expression in caspase-8-deficient cells. When the interferon-gamma-treated cells were treated with TRAIL, they underwent cell death, indicating restored sensitivity to TRAIL. Because most patients with Ewing’s sarcoma undergo chemotherapy, the investigators also looked at whether chemotherapy alters the levels of caspase-8 in Ewing’s sarcoma tumors. Using samples from the same 47 patients, they compared the number of tumor cells expressing caspase-8 between tumor samples collected before and after chemotherapy. They did not find any significant difference between the samples, suggesting that chemotherapy does not select for tumor cells that lack caspase-8, which would make subsequent treatment with TRAIL less effective. Data indicated that caspase-8 does not influence the sensitivity of Ewing’s sarcoma tumors to chemotherapy. The investigators confirmed these results in the laboratory. When cells with different levels of caspase-8 expression were treated with the chemotherapy drug doxorubicin, no differences in sensitivity to the drug were seen. Changing the levels of caspase-8 expressed by the cells, genetically or by adding interferon-gamma, did not alter their sensitivity to chemotherapy, indicating “that the combination of TRAIL and interferon-gamma with standard chemotherapeutics in Ewing’s sarcoma could be feasible,” stated the authors. “I’d like to see a clinical trial in humans of interferon-gamma and TRAIL or an agent that interacts with the TRAIL receptor, because I do believe that adding interferon-gamma will make cells more susceptible to TRAIL-induced killing,” said Dr. Mackall. An early phase study will be particularly important, she explained, because it is possible that adding interferon-gamma to treatment with TRAIL might also increase the incidence of side effects.
Cells That Cause Ewing's Sarcoma IdentifiedResearchers in France have discovered the cells that cause Ewing’s sarcoma. They are cells of the mesenchyme, the connective tissue that supports other tissues. The researchers have also succeeded "to make" the tumor cells to become virtually normal mesenchymal cells again. These results, published in the May 7 issue of Cancer Cell, open up new therapeutic possibilities for blocking the development of Ewing's sarcoma in young patients. Cancers rarely have a simple molecular signature-a specific mutation that causes tumor growth. In the case of Ewing's sarcoma, a molecular signature was identified and characterized in 1992 by Olivier Delattre's Inserm team at the Institut Curie. It is an accidental change of genetic material between two chromosomes, which results in the formation of a mutant gene, which codes for an abnormal protein called EWS/FLI-1. This discovery led on to the development of a diagnostic test for Ewing's sarcoma in 1994. Yet until now, the nature of the cell in which this mutation occurs was unknown. The group of Olivier Delattre, the Director of Inserm Unit 830 "Genetics and Biology of Cancer" at the Institut Curie, and the team of Pierre Charbord, the Director of Inserm Laboratory ERI5 "Microenvironment of Hematopoiesis and Stem Cells" in Tours, have now discovered that Ewing's sarcoma are caused by cells of the mesenchyme, a connective tissue that supports other tissues. They have shown that the profile of the transcriptome of Ewing's sarcoma resemble that of mesenchymal cells, particularly mesenchymal stem cells, when EWS/FLI-1 is inhibited.
By inhibiting the abnormal protein EWS/FLI-1 that causes Ewing's sarcoma, the researchers also "forced" the tumor cells to return to their original status of mesenchymal stem cells, which can then differentiate normally into bone or fat cells. This approach opens up new therapeutic prospects, since by forcing the cells to resume their original function it may be possible in the future to make them less aggressive and prevent their proliferation. As long as the tumor cells are still able to fulfill their function, they generally proliferate slowly, and the prognosis is good; once they lose this capacity, however, the tumor cells become highly aggressive. This discovery could allow the research group to produce an animal model of Ewing's sarcoma, an essential stage in the development of new treatments.
University of Pittsburgh Scientists Find New Contributor to Aggressive Cancers Mutations in the cell adhesion molecule known as integrin alpha 7 (integrin a7) lead to unchecked tumor cell proliferation and a significantly higher incidence in cancer spread, or metastasis, in several cancer cell lines, report researchers at the University of Pittsburgh School of Medicine in a study published in the Journal of the National Cancer Institute. These findings suggest that integrin a7 represents an important new target for cancer therapy and prevention. Integrin a7 belongs to a major class of cell membrane proteins that play a role in the attachment of a cell to the extracellular matrix (ECM), which is the material that holds cells within a particular type of tissue together. Integrins also help cells attach to one another and are involved in transmitting chemical signals between cells and the ECM.
In this study, the researchers, led by Jianhua Luo, M.D., Ph.D., associate professor in the division of molecular and cellular pathology, University of Pittsburgh School of Medicine, examined whether this gene is mutated in specimens of various human cancers as well as whether the level of integrin a7 expression is associated with clinical relapse of human cancers. They also investigated whether integrin a7 has tumor suppressor activity. To determine whether mutations in integrin a7 contribute to cancer, Dr. Luo and his collaborators sequenced the integrin a7 genes from 66 human cancer specimens and cell lines representing a number of different kinds of cancer, including cancer of the prostate, liver, brain (glioblastoma) and muscle (leiomyosarcoma). They found mutations in the integrin a7 gene, particularly those that resulted in an abnormally shortened protein product, or truncation, in 16 of 28 prostate cancers. They also found truncation-inducing mutations in five of 24 liver cancer samples, five of six glioblastomas, and one of four leiomyosarcomas.
Integrin a7 mutations also were associated with a significant increase in the recurrence of cancer among patients. Nine of 13 prostate cancer patients with integrin a7 mutations experienced a recurrence of their cancer after radical prostatectomy versus only one of eight prostate cancer patients without such mutations. There were five recurrences among eight hepatocellular carcinoma patients with integrin a7 mutations versus only one recurrence of cancer among 16 patients without such mutations.
To examine the effect of alterations in the level of integrin a7 on tumor formation, the researchers assessed the ability of cancer cells to form colonies in a standard growth medium after increasing or decreasing the level of normal integrin a7 in the cell lines. In this experiment, control cancer cells formed large colonies with up to 100 cells each. Cancer cells with normal levels of integrin a7 expression formed fewer and smaller colonies. When the investigators decreased the level of integrin a7 in two cancer cells lines using siRNAs, or silencing RNAs, both cell lines formed more colonies and grew better than corresponding control cell lines. “When we increased levels of normal integrin a7 in cancer cells, they grew at a much slower rate. This suggests that this protein is a fairly potent tumor suppressor,” said Dr. Luo.
Dr. Luo and his coworkers then investigated the role of integrin a7 in metastasis by examining the relationship between the level of integrin a7 expression and cell migration by increasing the expression of normal integrin a7 in three cell lines. The migration rate was significantly reduced in all of the cells compared to those in which the expression of integrin a7 remained deficient, suggesting that the level of normal integrin a7 expression is inversely associated with tumor cell migration.
Finally, to investigate whether normal integrin a7 possesses tumor suppressor activity, the researchers implanted human cancer cells into immune deficient mice. Some mice received tumor cells in which levels of integrin a7 were increased, others received tumor cells in which the levels of normal integrin a7 were decreased. Six weeks after mice were implanted with cancer cells in which levels of normal integrin a7 were deficient, they had tumors with an average volume about four times as large as mice with implanted cancer cells in which normal integrin a7 levels were increased. Similarly, the researchers found no visible metastasis in mice with tumors in which levels of normal integrin a7 had been increased. On the other hand, they did find evidence of metastasis in three of 12 mice with one type of tumor deficient in normal integrin a7 and in four of the 12 mice with another type of tumor deficient in normal integrin a7. Furthermore, the six-week survival of mice bearing tumors with increased levels of normal integrin a7 was higher than that of mice bearing tumors in which normal integrin a7 had not been experimentally increased. Thus, increasing the level of normal integrin a7 in tumors was associated with decreased tumor growth and metastasis in this animal model.
According to Dr. Luo and his coworkers, these findings suggest that not only is integrin a7 an important tumor suppressor, but it is potentially a critical new target for cancer treatment. “Our study shows rather definitively that when we experimentally decreased the level of integrin a7 protein or the protein was naturally mutated in cells, those cells lost their inhibitory signals for both cell migration and proliferation. This suggests that the loss of integrin a7 activity may lead to unchecked tumor cell proliferation and a significantly increased risk of tumor metastasis. More importantly, it suggests that if we can somehow restore normal integrin a7 levels in tumor cells in vivo, we may be able to reduce the risk of them spreading to other sites, which would be a significant achievement in cancer therapy,” explained Dr. Luo.
The Desmoid Tumor Research Foundation (DTRF) would like to: (1) notify researchers of its upcoming Request for Research Grant Proposals; and, (2) compile a comprehensive list of physicians experienced in desmoid tumors for its online physicians directory. If you would like to be included in either or both of the these lists, please email Jeanne Whiting Click here to see a related story about DTRF in this issue of ESUN.
HEM/ONC TODAY–Clinical News in Oncology and Hematology provides its 8th Annual Guide to cancer drugs in the pipeline and their developmental status as a downloadable PDF document. You can use the binoculars icon on the left hand side of the PDF to search for drugs in the pipeline for sarcomas, solid tumors, etc.
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