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In this JCO PO Article Insights episode, Fergus Keane provides a summary on "Microsatellite Instability Is Insufficiently Used as a Biomarker for Lynch Syndrome Testing in Clinical Practice", by Papadopoulou, et al published January 25, 2024 TRANSCRIPT The guest on this podcast episode has no disclosures to declare. Fergus Keane: Hello and welcome to JCO Precision Oncology Article Insights. I'm your host, Fergus Keane, an ASCO Editorial Fellow. Today, I will be providing a summary of the article entitled "Microsatellite Instability Is Insufficiently Used as a Biomarker for Lynch Syndrome Testing in Clinical Practice" by Dr. Eirini Papadopoulou. The mismatch repair pathway has gained interest in recent years due to advances in precision oncology, the widespread use of immune checkpoint blockade, and next-generation sequencing-based assays to identify microsatellite instability. The mismatch repair pathway has a key role in DNA repair and maintaining genomic stability. Tumor cells, which are MMR deficient are prone to mismatch errors in the microsatellite regions during DNA replication. Microsatellite instable, referred to as MSI-High tumors are observed across a variety of tumor types, most commonly colorectal and endometrial cancers.  Germline Lynch syndrome is caused by inactivating variants in one of five primary MMR genes, namely MSH2, MLH1, MSH6, PMS2, and EPCAM, and is associated with an autosomal dominant pattern of inheritance. Mismatch repair deficiency is observed in most tumors in individuals with Lynch syndrome and can also occur sporadically. In mismatch repair deficient colorectal cancer, sporadic cases are identified by BRAF V600E mutations or MLH1 gene promoter hypermethylation. The absence of both of these findings should raise suspicion for germline Lynch syndrome. The aim of this study was to report the prevalence of microsatellite instability in a large cohort of patients in Europe, specifically Greek patients. In addition, the authors aimed to evaluate the proportion of patients with microsatellite instability referred for germline testing and what factors appeared to influence clinician decision to refer for germline testing. 4553 patients with metastatic cancer were included between January 2020 and April 2023. All patients were referred for MSI analysis, and at physician discretion, BRAF V600E and MLH1 gene methylation analyses were available. Approximately half of patients included had colorectal cancer. 5.27% of patients exhibited MSI-High in total, of whom 58% were female and 42% were male.  The rates of MSI-High cancers varied according to tumor type, but the highest rates observed in patients with endometrial cancer at 15.69%, gastric cancer at 8.54%, colorectal cancer at 7.4%, and urinary tract cancers at 4.55%. Of the MSI-High patients, with colorectal cancer identified, 24.85% had a BRAF V600E. Excluding these patients, 198 were eligible for genetic testing with a hereditary cancer panel. Of these, only 22.7% were actually referred for a hereditary panel. The median age at diagnosis in this group was 59 years, compared with 66 years for those who were not referred for germline analyses. The age at diagnosis and referral for genetic analyses were significantly correlated.  Beyond colorectal cancer, patients with other cancer types who were also referred for germline testing included nine patients with endometrial cancer, four with gastric cancer, two with ovarian cancer, one with breast cancer, and one with gallbladder cancer, and referral patterns differed by tumor type. Of patients with colorectal and endometrial cancer, 24.4% had a positive germline mismatch repair variant identified. Of note, while the median age of patients with a pathogenic or likely pathogenic germline result was 48.5 years, over 40% of patients with a pathogenic germline result were aged over 50 years, highlighting that age alone should not be the only criterion for consideration of a referral for germline analysis in such patients.  This study highlights some important points. First, MSI analysis is an important biomarker for a variety of tumor types, including colorectal, gastric, and endometrial cancer, but also less commonly associated cancer types. Second, only 22.73% of patients eligible for germline analyses were referred in this patient cohort, despite recommendations from international guidelines indicating that a proportion of patients with a possible familial predisposition syndrome may not be tested for same. Third, key features such as age at diagnosis and tumor type influence the clinical decision for referral for genetic analysis.  The authors conclude that the value of MSI testing as a potential screening tool for identification of patients with a higher risk for germline pathogenic variants may be underappreciated and highlights this important biomarker. Thank you for listening to JCO Precision Oncology Article Insights, and please tune in for the next topic. Don't forget to give us a rating and review, and be sure to subscribe so that you don't miss an episode. You can find all ASCO shows at asco.org/podcasts The purpose of this podcast is to educate and to inform. This is not a substitute for professional medical care and is not intended for use in the diagnosis or treatment of individual conditions.   Guests on this podcast express their own opinions, experience, and conclusions. Guest statements on the podcast do not express the opinions of ASCO. The mention of any product, service, organization, activity, or therapy should not be construed as an ASCO endorsement.      

Drs Sumanta Pal and Rana McKay discuss the use of biomarkers in treating patients with renal cell carcinoma to identify whose disease will recur and who will respond to therapy. Relevant disclosures can be found with the episode show notes on Medscape (https://www.medscape.com/viewarticle/968743). The topics and discussions are planned, produced, and reviewed independently of advertiser. This podcast is intended only for US healthcare professionals. Resources Longitudinal Molecular Profiling of Circulating Tumor Cells in Metastatic Renal Cell Carcinoma https://ascopubs.org/doi/10.1200/JCO.22.00219 The Detection of EpCAM+ and EpCAM­– Circulating Tumor Cells https://www.nature.com/articles/srep12270 Optimized Management of Nivolumab and Ipilimumab in Advanced Renal Cell Carcinoma: A Response-Based Phase II Study (OMNIVORE) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768333/ ctDNA Guiding Adjuvant Immunotherapy in Urothelial Carcinoma https://www.nature.com/articles/s41586-021-03642-9 Clinical Activity and Molecular Correlates of Response to Atezolizumab Alone or in Combination With Bevacizumab Versus Sunitinib in Renal Cell Carcinoma https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721896/ Atezolizumab Plus Bevacizumab Versus Sunitinib in Patients With Previously Untreated Metastatic Renal Cell Carcinoma (IMmotion151): A Multicentre, Open-Label, Phase 3, Randomised Controlled Trial https://linkinghub.elsevier.com/retrieve/pii/S0140-6736(19)30723-8 Nivolumab Plus Ipilimumab Versus Sunitinib in Advanced Renal-Cell Carcinoma https://www.nejm.org/doi/full/10.1056/nejmoa1712126 Genetic Testing to Select Therapy for the Treatment of Advanced or Metastatic Kidney Cancer, OPTIC RCC Study https://clinicaltrials.gov/ct2/show/NCT05361720 Study of Cabozantinib in Combination With Nivolumab and Ipilimumab in Patients With Previously Untreated Advanced or Metastatic Renal Cell Carcinoma (COSMIC-313) https://clinicaltrials.gov/ct2/show/NCT03937219 Single-Cell RNA Sequencing of Human Kidney https://www.nature.com/articles/s41597-019-0351-8 Progressive Immune Dysfunction With Advancing Disease Stage in Renal Cell Carcinoma https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138872/

I designed the EPCAM strategy to help reduce the food waste each household produce. This strategy will prepare parents and caregivers on how well to collaborate with their children and family members on how to stop wasting food.

Vitamin A derivative selectively kills liver cancer stem cells  RIKEN Center for Integrative Medical Science (Japan), April 23, 2021 Acyclic retinoid, an artificial compound derived from vitamin A, has been found to prevent the recurrence of hepatocellular carcinoma (HCC), the most common form of liver cancer. Now, in research published in Proceedings of the National Academy of Sciences, scientists have discovered that the compound targets one class of cancer stem cells, preventing them from giving rise to new tumors. HCC is a highly lethal cancer, which causes approximately 600,000 deaths each year around the world, making it the second deadliest cancer after non-small cell lung cancer. One of the reasons for the high lethality is that it has a high rate of recurrence—surgery and other treatments are initially effective, but the cancer often relapses. As a result, researchers have looked for ways to prevent recurrence, and acyclic retinoid was recently found to be effective in stopping recurrence of tumors. However, scientists were not sure exactly why it worked. To find clues, a research group led by Soichi Kojima of the RIKEN Center for Integrative Medical Science looked at the transcriptome of cells that had been exposed to acyclic retinoid, and found that compared to control untreated cells, they had low expression of MYCN, a gene that is often expressed in tumors and is correlated with poor prognosis. Further experiments, which involved deliberately repressing the expression of the gene in cancer cells, showed that the reduction in MYCN expression led functionally to slower cell-cycle progression, proliferation, and colony formation, and to greater cell death, implying that the action of the acyclic retinoid on MYCN was slowing the cancer growth. The group then focused on the role of "cancer stem cells"—special cells that are able to survive the onslaught of chemotherapy or other treatments and to then differentiate into new cancer cells, leading to recurrence. They found, indeed, that high expression of MYCN was correlated with the expression of a number of markers that are associated with cancer stem cells. "The most interesting part of our finding," says Kojima, "is when we then looked at different subpopulations of heterogeneous cancer cells. We found one specific group of EpCAM-positive cancer stem cells, where MYCN was elevated. We wondered if perhaps the key to acyclic retinoid's effect was its ability to target these hepatic cancer stem cells." Indeed, experiments revealed that when exposed to acyclic retinoid, in a dose dependent manner, the EpCAM-positive cells were selectively depleted. To test whether this had clinical significance, they took liver biopsies of patients who had been given acyclic retinoid following liver cancer surgery, and found that in four of the six who had received a higher dosage of 600 mg/d but rather than 300 mg/d, there were decreased levels of MYCN expression, suggesting that MYCN expression in response to acyclic retinoid could be an important part of the difference in recurrence seen in trials. Finally, they looked at data from the Cancer Genome Atlas, and found that elevated expressionof MYCN correlated with dramatically poorer prognosis. According to Kojima, "It is remarkable that the acyclic retinoid clearly targets a certain category of cancer stem cells, and this provides us with important hints for decreasing cancer recurrence and truly curing patients. We are waiting to see what clinical data will show us." A phase 3 clinical trial of acyclic retinoid (also called Peretinoin), is currently underway in Korea, Taiwan and Singapore to test the drug's ability to prevent HCC recurrence.     Light up your mind: A novel light-based treatment for neurodegenerative diseases Researchers review growing knowledge on the methods and applications of light therapy in treating neurodegenerative diseases Soochow University (China), April 2021 A lot about the human brain and its intricacies continues to remain a mystery. With the advancement of neurobiology, the pathogenesis of several neurodegenerative diseases (ND) has been uncovered to a certain extent, along with molecular targets around which current therapies revolve. However, while the current treatments offer temporary symptomatic relief and slow down the course of the disease, they do not completely cure the condition and are often accompanied by a myriad of side effects that can impair normal daily functions of the patient. Light stimulation has been proposed as a promising therapeutic alternative for treating various ND like Alzheimer's disease (AD), Parkinson's disease (PD), cognitive and sleep disorders. Light therapy consists of controlled exposure to natural daylight or artificial light of specific wavelengths. While neurologists worldwide have begun testing its use in clinical practice, less remains understood about the mechanisms behind how light affects neurological function. Thus, in a review article now published in Chinese Medical Journal, researchers from China comprehensively summarize the growing knowledge on the mechanism of action, effectiveness, and clinical applications of LT in the treatment of ND. Neurologist and author Dr. Chun-Feng Liu explains how their work can advance our understanding of novel emerging therapies for ND. "While light therapy has been investigated in mental and sleep disorders, comprehensive knowledge on its use in neurodegenerative diseases in lacking. We therefore sought to shed light on the potential therapeutic methods and implications of light therapy," he states. Our body function is tuned to a circadian or day and night rhythm. The clock that controls this rhythm is housed in the hypothalamus region of the brain. The genes expressed in this region are crucial in maintaining the circadian rhythm. Thus, a malfunction of these genes can disrupt the rhythmic cycle. These defects have been associated with neurodegenerative, metabolic and sleep disorders. External stimuli such as light, physical activity and food intake can help reset the clock and restore normal circadian rhythms, thus alleviating symptoms. Another mechanism by which the clock controls circadian rhythms is through the secretion of the melatonin (MT) hormone. MT secreted by the pineal gland in the brain is known to control sleep patterns as it is secreted in higher amounts in the night than the day. Light stimulation in this case suppresses the secretion of MT during the day time and thus reduces drowsiness. Interestingly, different tissue and organs in the body may respond differentially to light stimulation. Furthermore, different biomolecules expressed in circulating immune cells and stem cells are sensitive to specific wavelengths of light and thus elicit different responses by promoting the secretion of neurotrophic factors that can rescue neuronal functions. Next, the researchers go on to discuss the application of light stimulation in specific neurodegenerative disorders. In case of AD, a progressive dementia, sleep disturbance has been associated with an increased expression of biomarkers that promote disease progression. Patients with AD often experience confusion, emotional distress and hyperactivity after dusk and through the night. Preliminary clinical studies on AD mouse models as well as patients with AD suggest that light stimulation helps restore memory and cognition and decreases the burden of the pathogenic amyloid-β protein in the brain. Furthermore, LT has been shown to improve sleep quality and duration in patients with sleep disorders while bright environments help reduce anxiety and aggressive behaviors in patients with dementia. In case of PD, patients suffer from motor impairment, tremors and posture imbalance and also display non-motor symptoms such as insomnia, depression and fatigue that can collectively impair their quality of life. While LT has been shown to decrease non-motor symptoms to some extent, evidences on its direct benefits towards motor-function however are limited. The use of LT in other neurodegenerative disorders is currently at preclinical stages and needs to be pursued further. Overall, LT offers a safe and cost-effective alternative for treatment of ND. Additional studies and large scale clinical trials in this direction can help establish its effectiveness as a potential therapeutic strategy. Explaining the long term clinical applications of LT, Dr. Liu says, "The light box or light therapy lamp will help improve the sleep quality of patients with sleep disorders. Light stimulation will also likely have therapeutic effects on neurodegenerative diseases and seasonal depression. Further studies are needed to elucidate its effectiveness." This review not only advances our understanding on how LT functions in resetting the circadian rhythm and associated neurological symptoms but also highlights its applications in routine clinical practice.   Bad to the bone: Hebrew University reveals impact of junk food on kids' skeletal development Study provides first comprehensive analysis for how junk foods impact skeletal development. Hebrew University of Jerusalem, April 19, 2021 A team of researchers from the Hebrew University of Jerusalem has proven the linkages between ultra-processed foods and reduced bone quality, unveiling the damage of these foods particularly for younger children in their developing years. The study, led by Professor Efrat Monsonego-Ornan and Dr. Janna Zaretsky from the Department of Biochemistry, Food Science and Nutrition at the University's Faculty of Agriculture, was published in the journal Bone Research and serves as the first comprehensive study of the effect of widely-available food products on skeleton development. Ultra-processed foods--aka, junk food--are food items products that undergo several stages of processing and contain non-dietary ingredients. They're popular with consumers because they are easily accessible, relatively inexpensive and ready to eat straight out of the package. The increasing prevalence of these products around the world has directly contributed to increased obesity and other mental and metabolic impacts on consumers of all ages.  Children tend to like junk food. As much as 70% percent of their caloric consumption are estimated to come from ultra-processed foods. While numerous studies have reflected on the overall negative impact of junk food, few have focused on its direct developmental effects on children, particularly young children. The Hebrew University study provides the first comprehensive analysis for how these foods impact skeletal development. The study surveyed lab rodents whose skeletons were in the post embryonic stages of growth. The rodents that were subjected to ultra-processed foods suffered from growth retardation and their bone strength was adversely affected. Under histological examination, the researchers detected high levels of cartilage buildup in the rodents' growth plates, the "engine" of bone growth. When subjected to additional tests of the rodent cells, the researchers found that the RNA genetic profiles of cartilage cells that had been subjected to junk food were showing characteristics of impaired bone development.  The team then sought to analyze how specific eating habits might impact bone development and replicated this kind of food intake for the rodents. "We divided the rodents' weekly nutritional intake--30% came from a 'controlled' diet, 70% from ultra-processed foods", shared Monsonego-Ornan. They found that the rodents experienced moderate damage to their bone density albeit there were fewer indications of cartilage buildup in their growth plates. "Our conclusion was that even in reduced amounts, the ultra-processed foods can have a definite negative impact on skeletal growth."  These findings are critical because children and adolescents consume these foods on a regular basis to the extent that 50 percent of American kids eat junk food each and every day. Monsonego-Ornan added. "when Carlos Monteiro, one of the world's leading experts on nutrition, said that there is no such thing as a healthy ultra-processed food, he was clearly right. Even if we reduce fats, carbs nitrates and other known harmful substances, these foods still possess their damaging attributes. Every part of the body is prone to this damage and certainly those systems that remain in the critical stages of development."   Results From The World's Largest Wellbeing Study Are In: Here's What We Know South Australian Health and Medical Research Institute, April 20, 2021 For decades, researchers have known that positive mental wellbeing seems to deliver significant improvements in physical health, development, and lifespan – which suggests looking after your mind and mental state is one of the most effective ways to care for the rest of your body as well. But what's the best way to actually promote personal mental wellbeing? In a new study led by scientists in Australia, researchers cast a wide net, analyzing data from almost 420 randomized trials employing different kinds of psychological interventions to help improve mental states of wellbeing. The results – a meta-analysis examining data from over 53,000 participants involved in hundreds of psychological experiments – is being billed as the world's largest study of its kind on wellbeing, giving perhaps the most comprehensive overview ever on how interventions can help towards a healthy mind and body. In the wake of the COVID-19 pandemic and the myriad hardships it has brought all over the world, new insights on how to successfully bolster mental states are in high demand. "During stressful and uncertain periods in our lives, pro-actively working on our mental health is crucial to help mitigate the risk of mental and physical illness," says mental health researcher Joep Van Agteren from the South Australian Health and Medical Research Institute (SAHMRI). "Our research suggests there are numerous psychological approaches people should experiment with to determine what works for them." In itself that might seem obvious, but as the researchers point out, up until now our awareness of psychological interventions' relative efficacy has been obstructed, given much research treats mental wellbeing and mental illness as different things, although they are overlapping concepts in some ways. Here, the researchers tried to take a broader view, looking at how a wide range of different types of psychological intervention can benefit mental wellbeing, irrespective of any particular theoretical foundation in psychology. Amongst the many forms of interventions included, two in particular stood out for their consistent associations with positive findings across trial cohorts: mindfulness-based interventions, and multi-component PPIs (positive psychological interventions), which package together a range of treatment methods and activities designed to cultivate positive feelings, behaviors, and thinking patterns. To a lesser extent, other interventions also appeared to deliver benefits, including acceptance and commitment therapy-based interventions, cognitive therapy, singular PPIs, and interventions focusing on reminiscence. While the effect sizes of these interventions are often moderate, the analysis here suggests they are linked with positive improvements in wellbeing in both clinical and non-clinical populations – but there's no quick fix, the researchers emphasize. "Just trying something once or twice isn't enough to have a measurable impact," says co-author Matthew Iasiello, a project coordinator at SAHMRI's Mental Health and Wellbeing program. "Regardless of what method people are trying out, they need to stick at it for weeks and months at a time for it to have a real effect." In their paper, the researchers make the same point in a different way. "Our moderator analysis indicated that improvement in mental wellbeing seems to be related to effort," the team writes. "While the review did not find a clear linear dose-response effect, with more exposure leading simply to better treatment outcomes, the results do indicate that more intense interventions seem to lead to more pronounced changes." Another insight by the researchers is that many kinds of psychological interventions can be done safely in volunteer groups or via online platforms, not requiring clinical appointments with professionals such as psychologists. With mental illness projected to become the largest contributor to disease by 2030, electing to look after yourself with these sorts of activities might not only benefit your own mental wellbeing and health – but the health of the health system too. "It is therefore potentially a cost-effective addition to current referral pathways and treatment methods," says clinical psychologist Michael Kyrios from Flinders University. "We need to take everyone's wellbeing seriously and ensure we're taking the necessary steps to improve mental and physical health so we can prevent future complications for ourselves and keep healthcare costs down." The findings are reported in Nature Human Behaviour.     The Stuff Beer Cans are Made from is Linked to Alzheimer's Disease Keele University (UK), April 15, 2021 There appears to be a troubling link between aluminum in the brain and the early signs of Alzheimer’s Disease, according to a new study. Researchers have known for years that aluminum has something to do with Alzheimer’s, but now Keele University scientists have discovered that the metal pops up at the same places in the brain as the tangles of tau protein that appear in the early stages of the disease, according to research published last month in the Journal of Alzheimer’s Disease Reports. The discovery suggests that it’s possible that aluminum could even play a role in forming those tangles and plaques — which precede the onset of Alzheimer’s — in the first place. “The presence of these tangles is associated with neuronal cell death, and observations of aluminum in these tangles may highlight a role for aluminum in their formation,” lead study author Matthew Bold said in a press release. That doesn’t mean that you need to ban aluminum cans from your home. Aluminum, perhaps introduced through food or other exposures, is commonly found in healthy brains, according to the Alzheimer’s Society, a dementia-focused charity based in London. But as people age, their kidneys may lose the ability to filter it out of the brain ­— potentially leading to the Alzheimer’s ties uncovered in the new study. “Aluminum accumulation has been associated with Alzheimer’s disease for nearly half a century,” Journal of Alzheimer’s Disease editor-in-chief George Perry said in the release, “but it is the meticulously specific studies of Drs. Mold and Exley that are defining the exact molecular interaction of aluminum and other multivalent metals that may be critical to formation of the pathology of Alzheimer’s disease.”     Drought-resistant cactus pear could become a sustainable food and fuel source, new research shows University of Nevada, April 16, 2021 Cactus pears could become a sustainable source of food and fuel in places in need of these two resources. Those are the findings of a recent study by researchers from the University of Nevada, Reno. Published in the journal GCB Bioenergy, the study covered five years of research. The group had set out to look at how successful different varieties of cactus pear would fare in warm, dry climates. They found that the prickly pear variety (Opuntia ficus-indica) produced the most fruit and used up 80 percent less water than other varieties to do so. With drought and heatwave events becoming more common worldwide, crops like corn and soybean may likely be heavily affected because they require more water than what might be available in the future. People will need to look for alternative crops that require less water, can tolerate droughts and still bear fruits. Cactus pears as sustainable food and fuel source Given current climate trends, the world is poised to get hotter and drier in the future. Therefore, plants that are drought-resistant and able to produce food with little water might soon become major sources of food. According to study co-author John Cushman, about 42 percent of all land on Earth is classified as arid or semi-arid. Therefore, there is enormous potential for planting cactuspears. Doing so has two main benefits. For starters, scientists can grow cactus pears in fields that are far too arid to be suitable for other crops. This increased production would put cactus pears on the map as food. Many cultures worldwide already eat the fruits from cactus pears and even the cactus pads themselves. However, cactus pears and other edible cactus varieties are far from being a major food and forage crop in the United States, let alone around the world. But that is a missed opportunity because cactus pear fruits can be used just like other fruits. They are especially great for making jams because they contain natural sugars. They can also be consumed fresh or pickled once the spines have been removed. They are also great for feeding livestock due to their high water content. The other benefit of utilizing arid fields for the cultivation of cactus pears is carbon sequestration. They capture and store carbon dioxide from the atmosphere, serving as a land-based carbon “sink.” They can also be harvested and used as raw materials for biofuels to replace fossil fuels. “That’s the benefit of this perennial crop,” explained Cushman. After you have harvested the fruits and pads for food, you will be left with a large amount of biomass that can be used for biofuel production, he said. (Related: Hemp: the versatile biofuel that could save America’s energy independence.) Cushman and his colleagues plan to continue researching cactus pears and their potential as sustainable fuel or foods. They plan to understand what it is about the genetic makeup of cactus pears that makes them so drought-resistant and use that information to make other crops more drought-resistant as well. Scientists have long been interested in the potential of cactus pears to serve as food and fuel. In 2015, a team of researchers from the United Kingdom suggested that water-efficient plants like cacti could be the key to providing sustainable bioenergy for the future. Plants like cacti carry out photosynthesis through a crassulacean acid metabolism (CAM) system. They grow on arid and semi-arid land with low or unpredictable rainfall, which can make conventional farming difficult. Arid and semi-arid lands are unproductive. But they can be put to good use by filling them with cacti and many other CAM plants that can capture and store carbon efficiently. The researchers said CAM plants like prickly pear could make a huge contribution to sustainable biogas production this way.   Yeast in kefir drink combats disease-causing bacteria Ben-Gurion University (Israel), April 17, 2021 People may have been producing and drinking kefir, a fermented milk drink that originated in Tibet and the North Caucasus, for thousands of years. People can make the sour, slightly effervescent brew by infusing milk with kefir grains, which are a starchy matrix containing a symbiotic community of lactic acid bacteria, acetic bacteria, and yeasts.  The drink has many reputed health benefits, which include lowering cholesterol, reducing inflammation, and exerting an antioxidant effect.  In common with other probiotics, kefir also has antimicrobial properties. However, scientists were unsure exactly how it inhibits the growth of disease-causing bacteria. Researchers at Ben-Gurion University of the Negev (BGU) in Be’er Sheva, Israel, have now discovered that a type of yeast in kefir called Kluyveromyces marxianus secretes a molecule that disrupts bacterial communication. Scientists already knew that plants and algae produce this substance, called tryptophol acetate, but this is the first time that they have found a yeast that makes it. They discovered that tryptophol acetate interferes with “quorum sensing” — a form of microbial communication — in several disease-causing bacteria.  In quorum sensingTrusted Source, bacteria release signaling molecules into their surroundings. When the molecules reach a particular concentration, they trigger changes in the expression of genes in bacteria of the same species. These changes allow disease-causing bacteria to coordinate their activity according to their numbers. This coordination is necessary for some bacteria to defend themselves or attack their hosts.  In some cases, when they reach a certain density, the microbes may come together to form a slimy, protective coating, or “biofilm,” on a surface.  Disease-causing bacteria In lab cultures, the researchers found that tryptophol acetate had an inhibitory effect over quorum sensing in several disease-causing bacteria, including some Gram-negativeTrusted Source bacteria. Some of the tested species were: Pseudomonas aeruginosa, which causes pneumonia when it infects the lungs.  S. enterica, which is responsible for food poisoning.  Staphylococcus aureus, which can trigger sepsis, among other life threatening infections. V. cholerae, which causes cholera. The research, which Ph.D. student Orit Malka led, appears in the journal BMC Microbiome. “These results are notable, since this is the first demonstration that virulence of human pathogenic bacteria can be mitigated by molecules secreted in probiotic milk products, such as yogurt or kefir,” says senior author Prof. Raz Jelinek. The scientists focused in particular on the effect of tryptophol acetate on V. cholerae. They found that the substance blocked quorum sensing in this bacteria and reduced its virulence. It did this by changing the expression of bacterial genes that control quorum sensing. The researchers write that this kind of interference in bacterial communication may be commonplace in complex environments where many different microorganisms live together, such as in probiotic food or the human gut.   Living near pesticide-treated farms raises risk of childhood brain tumors Cedars-Sinai Medical Center, April 15, 2021 Pregnant women living within 2.5 miles of agricultural lands treated with pesticides have a greater risk of their children developing central nervous system (CNS) tumors, according to a recent study. Published on Wednesday, March 31, in the Environmental Research journal, the study also revealed that the pregnant women did not have to be working in agriculture or in close contact with pesticides for health-harming exposures to occur. Study co-author Christina Lombardi, a public health researcher at Cedars-Sinai Medical Center in Los Angeles, said there are large numbers of pregnant women and children living close to pesticide-treated farmlands. Both mothers and children could experience adverse health effects from their proximity to those farmlands. The study is not the first to show that pesticide use poses a threat to pregnant women and their children. But it is unique in that it showed the specific pesticides linked to the development of different kinds of CNS tumors. Maternal exposure to pesticides linked to childhood tumors Experts have examined pesticide exposures as risk factors for the development of childhood brain cancers. But they have yet to assess the risk of developing childhood brain cancers from exposure to specific pesticides. (Related: California is going after another dangerous pesticide: Chlorpyrifos has been linked to brain damage.) To that end, Lombardi and her colleagues from the University of California, Los Angeles made use of the California Cancer Registry to identify cases of childhood CNS tumors in children below six years old. Overall, the researchers found 667 cases of CNS tumors in children below six. They matched each one with 20 controls to increase the statistical power of their findings. They then checked pesticide application records from the California Department of Pesticide Regulation‘s (CDPR) Pesticide Use Reporting (PUR) system to determine whether chemicals classified as possible carcinogens were used within 2.5 miles of the mothers’ homes at the time of the children’s births. Results showed that maternal exposure to certain pesticides heightened the risk of certain childhood CNS tumors by 2.5 times, even if the mother was not a farmworker. Pesticides found to increase the risk of childhood CNS tumors include thiophanate- and kresoxim-methyl, chlorothalonil, bromacil, triforine, propiconazole, dimethoate and linuron. Co-author Julia Heck said their findings are more precise than those of previous studies on pesticide exposure, which usually grouped pesticide use into broad categories based on type, such as herbicides or insecticides. Heck added that their results suggest that exposure to specific pesticides may best explain the results of earlier studies that reported a link between broader pesticide types and CNS tumors. Due to the risks that pesticide exposure poses on pregnant women and children, the researchers called for policy interventions to reduce pesticide exposure among people living near farms. “The simplest way to mitigate these risks is by reductions in exposure to pesticides,” said co-author Myles Cockburn. This can be done by restricting harmful practices like aerial spraying and air blast. Exposure to pesticides may also be reduced by promoting farming methods that limit reliance on pesticides.

I talk with Parvathy Krishnan, who discovered her Epcam mutation, as well as her husband's, after her children were diagnosed with CMMRD, constitutional mismatch repair deficiency.  We discuss Living with Lynch, and of course her son crashes the conversation when we discuss the Nets basketball team and Real Madrid soccer.  #rarediseaseday #livingwithlynch #sportsaremorethanjustagame 

March is colorectal cancer awareness day and March 22nd is Lynch Syndrome awareness day! Hereditary nonpolyposis colorectal cancer (HNPCC), more commonly known as Lynch syndrome, is an inherited condition that greatly increases the risk of many cancers, most notably colorectal cancer. This condition also increases the risk for other cancers such as endometrial, ovarian, and gastric cancer. Cancers that have a less severe risk increase include hepatobiliary tract, urinary tract, small bowel, pancreatic, brain/central nervous system, and sebaceous neoplasms. Not all people with Lynch syndrome have the same elevated risks. Risk levels depend on which gene(s) have mutations. Lynch syndrome is caused by mutations in the following genes, MLH1, MSH2, MSH6, PMS2, and/or EPCAM. This paper will explore the differing risks of types of cancers between genes along with how those gene variants are identified and measures that can be taken to reduce those risks. To learn more about Lynch Syndrome you can go to the Genetics Home Reference at ghr.nlm.nih.gov. Hear from Lynch syndrome patient, Georgia Hurst, who is also an advocate and has her own support group, "I Have Lynch Syndrome" on episode 25!

Thu, 18 Jun 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18614/ https://edoc.ub.uni-muenchen.de/18614/1/Henkel_Jonas.pdf Henkel, Jonas dd

Wed, 12 Nov 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18288/ https://edoc.ub.uni-muenchen.de/18288/1/Kremling_Heidi.pdf Kremling, Heidi ddc:610, ddc:600,

Thu, 9 Oct 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17544/ https://edoc.ub.uni-muenchen.de/17544/1/Hachmeister_Matthias.pdf Hachmeister, Matthias

Tue, 22 Jul 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17402/ https://edoc.ub.uni-muenchen.de/17402/1/Sarrach_Sannia.pdf Sarrach, Sannia ddc:610, ddc:600, M

Mon, 6 Jun 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13123/ https://edoc.ub.uni-muenchen.de/13123/1/Herrmann_Ines.pdf Herrmann, Ines ddc:570, ddc:500, Fakultät

Background: Peritoneal carcinomatosis (PC) is common in gastrointestinal (GI) cancer and there is no effective standard treatment. We investigated the tolerability and maximum tolerated dose (MTD) of the trifunctional antibody catumaxomab in patients with PC. Methods: In this open-label, phase I/II clinical trial, patients with epithelial cell adhesion molecule (EpCAM)-positive PC from GI cancer received 4 sequential intraperitoneal catumaxomab infusions: day 0: 10 mu g; day 3: 10 or 20 mu g; day 7: 30, 50, or 100 mu g; and day 10: 50, 100, or 200 mu g. Dose escalation was guided by dose-limiting toxicities. Results: The MTD was 10, 20, 50, and 200 mu g on days 0, 3, 7, and 10, respectively. Catumaxomab had an acceptable safety profile: Most common treatment-related adverse events (at the MTD) were fever, vomiting, and abdominal pain. At final examination, 11/17 evaluable patients (65%) were progression free: 1 patient had a complete and 3 a partial response. Median overall survival from the time of diagnosis of PC was 502 days. Conclusions: Intraperitoneal catumaxomab is a promising option for the treatment of PC from GI cancer.

Mon, 5 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12012/ https://edoc.ub.uni-muenchen.de/12012/1/Denzel_Sabine.pdf Denzel, Sabine ddc:570, ddc:500, Fakultät für Biolo

Fri, 26 Mar 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11463/ https://edoc.ub.uni-muenchen.de/11463/1/Gonzalez_Barbara.pdf González Carvajal, Bárbara

Die einzige Möglichkeit, Patienten mit Pankreaskarzinom zu heilen, besteht in einer möglichst frühzeitigen Diagnose und Operation. Nach einer kurativen Resektion versterben dennoch über 95% aller Patienten innerhalb der folgenden fünf Jahre an einem Rezidiv, was belegt, dass nach der Operation disseminierte Tumorzellen (DTZ) im Körper des Patienten verbleiben und deren Progression zu einem Rezidiv der Krebserkrankung führt. Beim Pankreaskarzinom wurde gezeigt, dass der Nachweis von DTZ mit dem Antikörper A45-B/B3 gegen Zytokeratin im Knochenmark und mit dem Antikörper Ber-EP4 gegen EpCAM im Lymphknoten mit einer schlechteren Prognose korreliert. Das Ziel der vorliegenden Dissertation war es, Einblicke in die Biologie von hämatogener und lymphogener Disseminierung beim Pankreaskarzinom zu erarbeiten. Zunächst wurde die Prävalenz von DTZ durch immunzytochemische Färbungen gegen die oben genannten epithelialen Marker bestimmt. Anschließend wurden die Zellen isoliert, ihre DNA amplifiziert und mit der komparativen genomischen Hybridisierung (CGH) auf numerische Aberrationen hin untersucht, welche mit den chromosomalen Veränderungen des Primärtumorkollektivs verglichen wurden. Diese Gegenüberstellung sollte ergründen, ob Ähnlichkeiten zwischen hämatogen disseminierten (H-DTZ), lymphogen disseminierten Tumorzellen (L-DTZ) sowie Primärtumoren (PT) bestehen und ob charakteristische Veränderungen für H-DTZ und L-DTZ existieren, welche möglicherweise im Rahmen der Disseminierung selektiert wurden. Die Prävalenz von DTZ bei Patienten mit nicht metastasiertem Pankreaskarzinom lag im Knochenmark bei 23,0% und im Lymphknoten bei 38,1%. Dieser Unterschied war nicht signifikant. Die genomische Analyse von DTZ und PT deckte auf, dass Primärtumore die höchste Anzahl an numerischen Aberrationen aufwiesen (Mittelwert: 12,8), gefolgt von den L-DTZ (Mittelwert: 9) und den H-DTZ (Mittelwert: 4,7). Zum anderen zeigten EpCAM-positive Zellen aus den Lymphknoten häufiger abnorme Karyotypen (78,3%) als Zytokeratin-positive Zellen aus dem Knochenmark (68,8%), während alle PT numerische Aberrationen aufwiesen. Die Häufigkeit von chromosomalen Gewinnen und Verlusten war unterschiedlich in den verschieden Gruppen und betrug bei H-DTZ 3,6 DNA-Gewinne und 1,1 DNA-Verluste, bei L-DTZ 4,5 DNA-Gewinne und 4,5 DNA-Verluste und bei PT 5,6 DNA-Gewinne und 7,2 DNA-Verluste pro analysierter Probe. Die Ähnlichkeitsanalyse numerischer Aberrationen der drei Kollektive zeigte, dass PT und insbesondere DTZ untereinander sehr heterogen sind. Charakteristische Veränderungen, die möglicherweise selektiert worden waren, wurden in erster Linie in PT gefunden, in geringerem Ausmaß auch in Tumorzellen aus Lymphknoten. Es handelte sich dabei in erster Linie um DNA-Verluste, wie zum Beispiel auf Chromosom 17 und 18, welche PT und L-DTZ gemeinsam waren und eine größere Ähnlichkeit zwischen diesen beiden Gruppen im Vergleich zu PT und H-DTZ nahelegen. Andererseits konnten auch Aberrationen identifiziert werden, die charakteristisch für die jeweilige Gruppe waren. Insbesondere handelte es sich dabei bei H-DTZ um DNA-Gewinne von 5p und 15q und bei L-DTZ um DNA-Gewinne von 4p oder 19 beziehungsweise DNA-Verluste von 11q oder 12p. Häufige DNA-Gewinne und -Verluste auf den Chromosomen 1, 3 oder 22 waren kennzeichnend für die Gruppe der Primärtumore. In der Analyse gepaarter Proben desselben Patienten waren gemeinsame Aberrationen zwischen PT und H-DTZ nur in der Hälfte (3/6), zwischen PT und L-DTZ dagegen in allen der untersuchten Fälle (2/2) anzutreffen. Auch die Anzahl dieser gemeinsamen Veränderungen war bei letzteren größer (Anteil gemeinsamer Veränderungen zwischen a) PT und H-DTZ: 5,9%, 5,9%, 4,8% und b) PT und L-DTZ: 16,3%, 8,9%). Der Vergleich solcher Aberrationen zwischen den verschiedenen Patienten mit gepaarten Proben dagegen führte nicht zur eindeutigen Identifikation typischer Veränderungen, welche im Zusammenhang mit einer hämatogenen oder lymphogenen Disseminierung gesehen werden können. Eine Zunahme numerischer Aberrationen mit fortschreitender Tumorprogression im Primärtumor wurde für die meisten soliden Tumoren nachgewiesen. Auch bei der Progression der pankreatischen intraepithelialen Neoplasie zum Pankreaskarzinom ist die Akkumulation von genetischen Veränderungen zu beobachten. Vor diesem Hintergrund deuten die vorliegenden Ergebnisse daraufhin, dass die Disseminierung ein frühes Ereignis im Rahmen der Progression des Pankreaskarzinoms ist. In sehr frühen Tumorstadien, welche möglicherweise von den Genomprofilen der DTZ im Knochenmark repräsentiert werden, sind in erster Linie DNA-Gewinne anzutreffen. Interessanterweise scheinen diese DTZ im Milieu des Knochenmarks keine Deletionen zu akkumulieren. Mit fortschreitender Tumorprogression kommt dagegen DNA-Verlusten eine zunehmend bedeutende Rolle zu, was in DTZ aus Lymphknoten sowie im Primärtumor zu beobachten ist. Diese Beobachtungen bilden die Grundlage für eine Vielzahl weiterführender Fragestellungen, wie zum Beispiel die Identifikation der genetischen Veränderungen, die am Anfang der genetischen Progression stehen. Gerade diese Veränderungen könnten von allen Tumorzellen geteilt werden und somit ideale Zielstrukturen für neue Therapieansätze darstellen.

Background: Epithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety. Methods: We recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation. Results: ING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fc gamma 1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells. Conclusion: A moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.

Mon, 14 Dec 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/10947/ https://edoc.ub.uni-muenchen.de/10947/1/Amann_Maria.pdf Amann, Maria ddc:570, ddc:50

Über die Hälfte aller malignen Pleuraergüsse sind durch solide Tumore des Mamma- und Bronchialkarzinoms bedingt und stellen eine Fernmetastasierung dar. Trotz therapeutischer Maßnahmen wie der Pleurodese mit Talkum ist die Prognose der Patienten schlecht und ihre mittlere Überlebenszeit beträgt nur wenige Monate. Deswegen sind innovative und effiziente Therapiestrategien für die Therapie der Pleurakarzinose dringend notwendig. In dieser Arbeit wurden maligne Pleuraergüsse (MPE) von Mamma- und Bronchialkarzinompatienten auf zellulärer und molekularer Ebene charakterisiert. Die zelluläre Zusammensetzung der malignen Pleuraergüsse und die Expression prognose-, therapie- und chemoresistenzassoziierter Antigene wurden mittels immunhistochemischer Methoden nachgewiesen. Die Charakterisierung der Ergüsse zeigte eine patienten- und tumorbedingte zelluläre und molekulare Heterogenität. Dabei unterschieden sich die malignen Pleuraergüsse bezüglich der Ergussmenge, der aus den Ergüssen isolierten Zellzahl, ihrer zellulären Zusammensetzung, der Beschaffenheit der Pleurakarzinomzellen (Einzelzellen, Aggregate) und ihrer Antigenexpression. Die Analyse des Hormonrezeptorstatus ergab einen signifikanten Verlust des Östrogen- (p=0,002) und Progesteronrezeptors (p=0,0005) auf den Pleurakarzinomzellen im Vergleich zum korrespondierenden Primärtumor. Therapierelevante Antigene aber wie z.B. das epitheliale Antigen EpCAM, das Glykoprotein MUC-1 und der Wachstumsfaktorrezeptor EGF-R waren auf mehr als 70 % der MPE beim Mamma- und Bronchialkarzinom vertreten. In dieser Arbeit wurde daraufhin in einer ex vivo Studie die Wirksamkeit des bispezifischen Antikörpers MT110 gegen das Antigen EpCAM und den T-Zellrezeptor CD3 als Therapiestrategie für die Behandlung maligner Pleuraergüsse gestestet. Der Anteil der antikörpervermittelnden spezifischen Lyse der EpCAM-positiven Pleurakarzinomzellen wurde mit Hilfe der Durchflußzytometrie (FACS) bestimmt. Die MT110 abhängige Aktivierung der CD4- und CD8-positiven T-Zellen wurde über die Antigene CD25, Granzym B und die Ausschüttung immunmodulierender Zytokine nachgewiesen (FACS). Die ex vivo Therapie der MPE beim Mammakarzinom ergab eine dosisabhängige signifikante spezifische Lyse der Targetzellen nach 48 h und 72 h mit 10 ng/ml und 1000 ng/ml MT110 (48 h, p = 0,03; 72 h, p = 0,016). Bei einer Behandlungsdauer von 72 h erzielte MT110 in einer Konzentration von 1000 ng/ml eine spezifische Lyse der EpCAM-positiven Zellen von 57 % ± 29.5 % (MW ± stabwn). Die ex vivo Therapie der MPE beim Bronchialkarzinom führte zu einer durchschnittlichen spezifischen Lyse von 30 ± 38 %, die nicht signifikant war (72h, 1000 ng/ml). Der späte Aktivierungsmarker CD25 war nach 48h und 72h mit 1000 ng/ml MT110 auf den CD4- und CD8-positiven Pleuraergusszellen der Mammakarzinompatienten (p=0,03; p=0,016) und Bronchialkarzinompatienten (nicht signifikant) verstärkt exprimiert. Der erhöhte Nachweis von TNF-α (p = 0,016) und IFN-γ (p = 0,03) in den Mediumüberständen der MPE beim Mammakarzinom deutete auf eine TH-1 vermittelte Immunantwort hin. Die ex vivo Therapie der MPE mit MT110 zeigte, dass das epitheliale Antigen EpCAM ein geeignetes Zielantigen für die molekulare Therapie der Pleurakarzinose darstellt. Der bispezifische Antikörper MT110 bewirkte eine zielgerichtete spezifische Lyse der Pleurakarzinomzellen durch die Stimulation der autologen Immunzellen im malignen Erguss. Mögliche Einflüsse auf die Höhe der antikörperspezifischen Lyse sind das Vorhandensein von Aggregaten im Erguss, das Verhältnis der Effektor- und Targetzellen, sowie die Stimulierbarkeit der T-Lymphozyten. Die Wirkungsweise des Antikörpers MT110 muss in Zukunft für ein größeres Patientenkollektiv getestet werden. Der Antikörper befindet sich seit April 2008 in einer klinischen Phase I und wird bei Patienten mit EpCAM-positiven, lokal fortgeschrittenen, rezidivierten oder metastasierten Karzinomen auf Verträglichkeit und Antitumoraktivität hin untersucht. Zukünftige klinische Studien sind nötig, um die Wirksamkeit von MT110 bei Patientinnen mit MPE beim Mamma- und Bronchialkarzinom zu bestätigen.

Background: Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is frequently over-expressed in simple epithelia, progenitors, embryonic and tissue stem cells, carcinoma and cancer-initiating cells. Besides functioning as a homophilic adhesion protein, EpCAM is an oncogenic receptor that requires regulated intramembrane proteolysis for activation of its signal transduction capacity. Upon cleavage, the extracellular domain EpEX is released as a soluble ligand while the intracellular domain EpICD translocates into the cytoplasm and eventually into the nucleus in combination with four-and-a-half LIM domains protein 2 (FHL2) and beta-catenin, and drives cell proliferation. Methods: EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were investigated under varying density conditions using confocal laser scanning microscopy, immunoblotting, cell counting, and conditional cell systems. Results: EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were dependent on adequate cell-to-cell contact. If cell-to-cell contact was prohibited EpCAM did not provide growth advantages. If cells were allowed to undergo contact to each other, EpCAM transmitted proliferation signals based on signal transduction-related cleavage processes. Accordingly, the pre-cleaved version EpICD was not dependent on cell-to-cell contact in order to induce c-myc and cell proliferation, but necessitated nuclear translocation. For the case of contact-inhibited cells, although cleavage of EpCAM occurred, nuclear translocation of EpICD was reduced, as were EpCAM effects. Conclusion: Activation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine).

Mon, 16 Jun 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/8663/ https://edoc.ub.uni-muenchen.de/8663/1/Maetzel_Dorothea.pdf Maetzel, Dorothea ddc:500, ddc:570, Fakultät für Biologie

Plattenepithelkarzinome des Kopf-Hals-Bereichs (HNSCC) haben, vor allem im fortgeschrittenen Stadium, wegen einer hohen Rate an lokalen Lymphknotenmetastasen und Fernmetastasen eine schlechte Prognose. Aus diesem Grunde ist man auf der Suche nach effektiven adjuvanten Therapiemöglichkeiten. Das Prinzip der Radioimmuntherapie scheint hierfür sehr viel versprechend zu sein. Plattenepithelkarzinome des Kopf-Hals-Bereichs exprimieren die tumorassoziierten Antigene EpCAM und CK8. Ziel dieser Arbeit war es, die Biodistribution und die therapeutische Wirksamkeit der an 131I bzw. 123I gekoppelten Antikörper gegen EpCAM und Cytokeratin 8 (C215 und HK-8) in vivo im SCID-Mausmodell zu untersuchen. Zunächst wurden Studien zur Biodistribution durchgeführt. Zu definierten Zeitpunkten nach i.v.-Applikation von 131I-markierten IgG2a-Antikörper C215 bzw. HK-8 wurden Mäuse euthanasiert und der prozentuale Anteil der injizierten Dosis pro Gramm Gewebe (%ID/g) in den einzelnen Organen sowie im Tumor bestimmt. Szintigraphiestudien mit 123I-C215 und 123I-HK-8 ergaben ebenfalls Aufschlüsse über die systemische Verteilung der Radioimmunkonjugate (RIK). Zudem wurde die therapeutische Wirksamkeit von 131I-C215 untersucht. Hierzu wurden den xenotransplantierten Mäusen 25, 15 oder 5 MBq des RIK systemisch verabreicht. Die Kontrollgruppen erhielten entweder den unmarkierte Antikörper oder sterile Kochsalzlösung. In den Untersuchungen zur Biodistribution und in der Szintigraphie ließ sich eine spezifische Anreicherung der markierten Antikörper im Tumor nachweisen. Die erreichten Tumor/Nicht-Tumor- (T/NT-) Verhältnisse waren bei Vergleich beider RIK ähnlich. Durch Anstieg der T/NT-Verhältnisse nahezu aller Proben, konnte in beiden Fällen eine stetige Zunahme der RIK-Anreicherung im Tumor nachgewiesen werden. Das Nicht-Blut/Blut-Verhältnis zeigte ebenfalls sowohl für 131I-CK8 als auch 131I-C215 eine spezifische Anreicherung im Tumor. Das Problem eines relativ hohen Anteils beider RIK in Milz und Leber konnte durch zusätzliche Applikation von unspezifischem CD20-IgG2a verhindert werden, da dieser Effekt unter anderem auf einen Mangel an endogenem IgG2a in SCID-Mäusen zurückzuführen ist. In der Therapiestudie zeigten die Mäuse, denen 15 MBq injiziert worden waren, 24 Tage p.i. ein im Vergleich zum Applikationszeitpunkt relatives Tumorvolumen von 0,38. Bei den Mäusen, die 25 MBq erhalten hatten war an Tag 17 p.i. ein relatives Tumorvolumen von 0,56 zu verzeichnen. Es war also zu einer deutlichen Reduktion des Tumorvolumens um 44 bzw. 62 Prozent gekommen. Dies konnte jedoch nur auf Kosten einer hohen Toxizität erreicht werden. In der Therapiegruppe, die 5 MBq erhalten hatte und in den beiden Kontrollgruppen (NaCl oder unmarkierter Antikörper) war kein antitumoraler Effekt festzustellen. Hier zeigte sich erwartungsgemäß ein exponentielles Tumorwachstum. Die vorliegenden Ergebnisse lassen darauf schließen, dass Antikörper gegen EpCAM gut geeignet sind für eine radioimmuntherapeutische Behandlung von HNSCC, da die applizierten Antikörper in ausreichender Menge ihr Target, d. h. die Tumorzellen erreichten. Allerdings erwiesen sich die gewählten Dosierungen von 15 und 25 MBq 131I-C215 bei tumortragenden Mäusen als radiotoxisch.

Das epitheliale Zelladhäsionsmolekül EpCAM ist in der Tumorentstehung von Plattenepithelkarzinomen über- oder de novo exprimiert. Zudem korreliert die EpCAM-Expression in Tumorzellen positiv mit Proliferation und Entdifferenzierung. Es wurde in Vorarbeiten ein 1100 bp epcam-Promotorfragment kloniert, das spezifisch in EpCAM-positiven Zellen transkriptionell aktiv ist und durch TNFα in der Promotoraktivität reprimiert wird. In meiner Arbeit untersuchte ich, ob das 1100 bp epcam-Promotorfragment zur gezielten heterologen Genexpression geeignet ist. Zu diesem Zweck wurden drei Proteine ausgewählt: Grünes Fluoreszenz Protein (GFP), TNF receptor associated death domain Protein (TRADD) und Herpes Simplex Virus 1 Thymidinkinase (HSV1-TK). GFP diente der Visualisierung der Promotoraktivität im Fluoreszenzmikroskop. TRADD sollte die Apoptose in EpCAM-positiven Tumorzellen induzieren. Mit Hilfe der spezifischen Expression der HSV1-TK in EpCAM-positiven Zellen sollten Tumorzellen für Ganciclovir sensitiviert werden. Eine Therapie mit Ganciclovir sollte das Absterben der Tumorzellen bewirken. Die heterologe Genexpression wurde an einem zellulären Modellsystem von EpCAM-positiven und EpCAM-negativen HEK293 Zellen getestet. Dabei zeigten EpCAM-positive Zellen eine deutliche GFP-Expression, während EpCAM-negative Zellen sporadisch eine minimale Fluoreszenzintensität aufwiesen. Die EpCAM-spezifische Expression von GFP konnte im Immunoblot bestätigt werden. Um den Zusammenhang zwischen EpCAM- und GFP-Expression zu veranschaulichen, wurden die Ergebnisse der durchflusszytometrischen Messungen der EpCAM-Oberflächenexpression mit der GFP-Fluoreszenz verglichen. Damit konnte im zellulären Modellsystem von EpCAM-positiven und EpCAM-negativen HEK293 Zellen gezeigt werden, dass die epacm-Promotoraktivität zu einer heterologen Genexpression von GFP führt. Das zelluläre Modellsystem von EpCAM-positiven und EpCAM-negativen HEK293 Zellen wurde auf die Expression weiterer funktioneller Gene untersucht. Für das Funktionsgen TRADD konnte dabei weder eine EpCAM-spezifische heterologe Genexpression in der RT-PCR noch im Immunoblot nachgewiesen werden. In beiden Untersuchungen führten die Positivkontrollen zu einem Nachweis von TRADD. Da TRADD über komplexe Signalwege zur Bildung von TNFα führen kann, findet möglicherweise eine Inaktivierung des epcam-Promotors durch TNFα statt. Die heterologe Genexpression von HSV1-TK unter der Kontrolle des epcam-Promotors konnte im zellulären Modellsystem in der RT-PCR nachgewiesen und auf die EpCAM-positive Tumorzelllinie SKBR3 übertragen werden. Durch die Genexpression von HSV1-TK wurden EpCAM-positive HEK293 Transfektanten sensitiv gegenüber einer Behandlung mit Ganciclovir und zeigten eine deutlich reduzierte metabolische Aktivität im MTT-Ansatz bei Ganciclovirgabe. Dabei gewonnene Erkenntnisse wurden an der EpCAM-positiven Tumorzellinie SKBR3 bestätigt. Zusammengefasst konnte gezeigt werde, dass die heterologe Genexpression von HSV1-TK unter der Kontrolle des epcam-Promotors zu Ganciclovirsensitivität in EpCAM-positiven Zellen führte, jedoch nicht in EpCAM-negativen Zellen. Somit ist es denkbar, das 1100 bp epcam-Promotorfragment für die therapeutische Genexpression letaler Gene zur Elimination EpCAM-positiver Tumorzellen zu verwenden.

Das Epithelial Cell Adhesion Molecule, kurz EpCAM, ist ein transmembranes Zelladhäsionsmolekül, dessen Expression mit einer erhöhten Proliferation epithelialer Zellen einhergeht. Die veränderten proliferativen Eigenschaften werden durch eine EpCAM-vermittelte Signaltransduktion hervorgerufen, die u.a. die Transkription des Proto-Onkogens c-myc induziert. In dieser Arbeit konnte gezeigt werden, dass EpCAM die Transkription weiterer „down stream“-Zielmoleküle reguliert. Die mRNA-Synthese des Transkriptions-faktors Egr-1 wird durch EpCAM reprimiert. Dagegen induziert EpCAM die Transkription der Gene für RhoB und Calmodulin I. Außerdem korreliert in unterschiedlichen Karzinomzelllinien die native EpCAM-Expression mit der Transkriptionsrate von Calmodulin I. Zur Identifikation intrazellulärer Interaktionspartner wurde mit der kurzen zytoplasmatischen Domäne (26 Aminosäuren) von EpCAM ein Two-Hybrid Screening in Hefen durchgeführt. Es konnten 17 potentielle Interaktionspartner aus einer humanen cDNA-Bank identifiziert werden. Für die zwei Signaltransduktions-moleküle FHL2 und Cyclophilin A war es möglich, eine Interaktion mit EpCAM in humanen Zellen nachzuweisen. Darüberhinaus konnte gezeigt werden, dass die C-terminale LIM-Domäne von FHL2 für eine Assoziation mit EpCAM notwendig ist. Somit konnte in dieser Arbeit die Grundlage für eine weitere Analyse der EpCAM-vermittelten Signaltransduktion geschaffen werden.

Background: Gastric carcinoma is one of the most frequent cancers worldwide. Patients with gastric cancer at an advanced disease stage have a poor prognosis, due to the limited efficacy of available therapies. Therefore, the development of new therapies, like immunotherapy for the treatment of gastric cancer is of utmost importance. Since the usability of existing preclinical models for the evaluation of immunotherapies for gastric adenocarcinomas is limited, the goal of the present study was to establish murine in vivo models which allow the stepwise improvement of immunotherapies for gastric cancer. Methods: Since no murine gastric adenocarcinoma cell lines are available we established four cell lines (424GC, mGC3, mGC5, mGC8) from spontaneously developing tumors of CEA424/SV40 T antigen (CEA424/Tag) mice and three cell lines derived from double-transgenic offsprings of CEA424/Tag mice mated with human carcinoembryonic antigen (CEA)-transgenic (CEA424/TagCEA) mice (mGC2(CEA), mGC4(CEA), mGC11(CEA)). CEA424/Tag is a transgenic C57BL/6 mouse strain harboring the Tag under the control of a -424/-8 bp CEA gene promoter which leads to the development of invasive adenocarcinoma in the glandular stomach. Tumor cell lines established from CEA424/Tag- CEA mice express the well defined tumor antigen CEA under the control of its natural regulatory elements. Results: The epithelial origin of the tumor cells was proven by morphological criteria including the presence of mucin within the cells and the expression of the cell adhesion molecules EpCAM and CEACAM1. All cell lines consistently express the transgenes CEA and/or Tag and MHC class I molecules leading to their susceptibility to lysis by Tag-specific CTL in vitro. Despite the presentation of CTL-epitopes derived from the transgene products the tumor cell lines were tumorigenic when grafted into C57BL/6, CEA424/Tag or CEA424/Tag- CEA-transgenic hosts and no significant differences in tumor take and tumor growth were observed in the different hosts. Although no spontaneous tumor rejection was observed, vaccination of C57BL/6 mice with lysates from gastric carcinoma cell lines protected C57BL/6 mice from tumor challenge, demonstrating the tumorigenicity of the tumor cell lines in nontransgenic mice of the H-2(b) haplotype. Conclusion: These tumor cell lines grafted in different syngeneic hosts should prove to be very useful to optimize immunotherapy regimens to be finally tested in transgenic animals developing primary gastric carcinomas.

Im Rahmen der Arbeit wurde ein zelluläres System etabliert mit dem die transkriptionelle Aktivität des EpCAM-Promotors, ausgehend von der bisherig bekannten Promotorsequenz, beurteilbar ist. Experimentelle Basis war der Luziferase-Assay für den ein Luziferase-Reporterplasmid unter transkriptioneller Kontrolle der bisher bekannten EpCAM-Promotorsequenz generiert wurde. Es konnte mit Hilfe von FACS-Analysen und Immunoblots gezeigt werden, daß sich die gemessene transkriptionelle Aktivität in der tatsächlichen EpCAM-Expression widerspiegelt. Desweiteren konnte gezeigt werden, daß die kanzeromodulierenden Substanzen TNFalpha und TPA auf den EpCAM-Promotor reprimierend wirken. Im Falle von TNFalpha konnte experimentell belegt werden, daß die Repression über die spezifische Signaltransduktion über den NF-kappa-B-Signalweg zustande kommt, in dem letztlich NF-kappa-B über eine Kompetition um den wichtigen Transkriptionskoaktivator p300/CBP eine Repression des EpCAM-Promotors vermittelt.

Non-small cell lung cancer (NSCLC) is characterised by early dissemination of tumor cells resulting in a strikingly reduced overall survival despite complete surgical removal of the primary tumour. It has been suggested that proteases contribute to this apparent aggressiveness of lung cancer because they are involved in a variety of mechanisms associated with tumor progression such as invasion, migration and angiogenesis. To investigate the significance of protease expression and to identify potentially co-regulated molecules during early dissemination and in minimal residual disease, we performed cDNA array analysis of single disseminated cancer cells or small cell clusters isolated from bone marrow and lymph nodes of NSCLC patients. We obtained macroscopically tumor free lymph nodes and bone marrow aspirates from patients with operable NSCLC and enriched single disseminated cancer cells and small cell clusters by density gradient centrifugation. Subsequently, the freshly prepared cell suspensions were stained with an antibody against the epithelial surface molecule EpCAM and single positive tumour cells were isolated by micromanipulation. After global amplification of the single cell cDNA and non-radioactive labelling proteinase expression was assessed using a cDNA array consisting of several matrix metalloproteases, cathepsins, caspases, kallikreins and other serine / cysteine proteases. Until now we could isolate 46 EpCAM+ cells from 72 lymph nodes and 19 EpCAM+ cells from 71 bone marrow aspirates. For 30 cells the epithelial origin could be confirmed by the co-expression of several epithelial markers or by expression of the tumor specific MAGE antigens. Hybridisation of these cells on our protease cDNA array revealed the expression of various proteases in single cells and small cell clusters. Particularly, serine proteases, cathepsins and other cysteine proteinases are frequently expressed, while, contrary to our expectations, the transcripts of matrix metalloproteases (MMP) were rarely detected in the analysed cells.

Das epitheliale Adhäsionsmolekül EpCAM wird insbesondere in gesunden Adenoepithelien exprimiert, nicht aber in den mukösen Plattenepithelien der oberen Atem- und Verdauungswege. Aus diesen Geweben hervorgehende Plattenepithelkarzinome dagegen zeigen eine Überexpression von EpCAM. Die Ergebnisse der vorliegenden Arbeit zeigen zudem, dass die Plattenepithelkarzinome der Kopf-Hals-Region EpCAM außerdem in einer hyperglykosilierten Proteinvariante exprimieren, die das Ergebnis posttranskriptioneller Modifikation ist. So konnte gezeigt werden, dass in gesundem und in malignem Gewebe tatsächlich unterschiedliche Glykosilierungsformen von EpCAM vorliegen: In gesundem Gewebe wird EpCAM als unglykosiliertes Protein von 37 kDa exprimiert. Das aus Tumorgewebe stammende EpCAM hingegen war in den untersuchten Karzinomzelllinien aus SCCHN, Mamma- und Kolon und in den Gewebsproben aus Patienten mit SCCHN und mit Kolonkarzinomen hyperglykosiliert. Dabei zeigten die Karzinome ein heterogenes Bild mit unterschiedlichen Variationen und Kombinationen der verschiedenen Glykosilierungsformen. Gesundes EpCAM-positives Schilddrüsenepithel und auch gesunde Kolon- und Magenschleimhaut dagegen exprimierten vorwiegend unglykosilierte Formen von EpCAM. Innerhalb von Probenpaaren aus SCCHN und autologem Schilddrüsengewebe war EpCAM in den meisten Fällen (81,5 %) in der Karzinomprobe stärker glykosiliert als im gesunden Epithel, eine analoge Verteilung der Glykosilierungsmuster zeigten die Probenpaaren aus gesunder und neoplastisch veränderter Kolon- und Magenschleimhaut. In epithelialen Neoplasien wird EpCAM eine essentielle Bedeutung für die Karzinogenese zugeschrieben. Gleichzeitig dominieren in SCCHN und in anderen Karzinomen hyperglykosilierte Proteinvarianten von EpCAM, die in gesunden Epithelien nicht oder kaum anzutreffen sind. Man könnte daher vermuten, dass zwischen der posttranslationalen Hyperglykosilierung von EpCAM und den funktionellen Eigenschaften des tumorassoziierten Antigens in Karzinomzellen ein enger Zusammenhang besteht. Weitere Untersuchungen wären notwendig, um die molekulare Struktur und funktionelle Bedeutung der hyperglykosilierten Proteinvarianten von EpCAM in Karzinomen zu klären. Die Identifizierung karzinomspezifischer Epitope auf dem Glykoprotein EpCAM wäre Voraussetzung für die Entwicklung spezifischer Antikörpern, die hyperglykosiliertes EpCAM auf Karzinomzellen erkennen. Ziel wäre eine Immuntherapie mit EpCAM von hoher Karzinomspezifität und somit ein hoher therapeutischer Wirkungsgrad bei geringerer Toxizität. Für Patienten mit SCCHN könnte dies insbesondere bei minimal residual disease zu einer Verbesserung der bisher ungünstigen Prognose führen.

Fri, 30 Aug 2002 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/539/ https://edoc.ub.uni-muenchen.de/539/1/Muenz_Markus.pdf Münz, Markus ddc:570, ddc:500