The following is a presentation from the SELECTSCIENCE Cancer Research Summit 2023 (on Nov. 14. 2023): exploring the latest developments in cancer research with fellow scientists, manufacturers, and regulatory experts
Introduction:
SelectScience® Cancer Research Summit is celebrating its third year, bringing together the scientific community and pioneering manufacturers to foster collaboration and facilitate the dissemination of crucial updates in cancer research. Each year, scientific presentations by expert speakers deliver the latest findings in their respective topics, offering practical skills through engaging technology showcases, interactive resources by industry leaders- helping the community to harness the power of networking to drive impactful collaborations.
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For the second year in a row, ICRS top clinical advocate Dr. Noelle Cutter was elected to be one of the headliners of this global (virtual) medical event. With her, she brought fellow cancer researchers and two powerhouse publishers in women's health- Dr. Robert L. Bard (Sr. Cancer Imaging Radiologist) and Dr. Roberta Kline (OBGYN/ Genomics Expert). Together, Dr. Cutter's presentation promises to empower the medical community with the most comprehensive report on "Molecular Changes in Dense Breast Tissue" and its metabolic correlation with Breast Cancer.
This video presentation is comprised of 3 presentations which can be accessed in full assembly or as separate clips: (1) Dr. Noelle Cutter -part 1 (2) Dr. Robert Bard (3) Dr. Roberta Kline (4) Dr. Noelle Cutter - Part 2. Scroll below for direct access to all presentations and transcripts.
TRANSCRIPT & VISUAL PRESENTATION
By: Dr. Noelle Cutter (Part 1)
Hi everybody. I'm coming to you from Molloy University where in my lab, we study molecular changes in breast cancer. Joining me today, I have Dr. Robert Bard and Dr. Roberta Kline. Dr. Bard is an internationally recognized leader in the field of 21st century 3D ultrasonic volumetric doppler imaging. Dr. Bard specializes in advanced 3D sonography to detect cancers in numerous organs including the breast. Also with us is Dr. Roberta Kline, a board certified OB GYN Air Force veteran and a functional genomics and mind-body expert. She bridges conventional and alternative worlds to harness the best technologies and knowledge to create better health. 
The complexity of cancer can be reduced to a small number of underlying principles, all share common traits or hallmarks that govern transformation of normal cells to malignant cells. The famous Hanahan and we Weinberg paper was redesigned since its first publication back in 2001. The latest publication in 2022 included non mutational, epigenetic reprogramming and global changes in the epigenetic landscape are now recognized as a feature of many cancers.
Breast cancer is the most common cancer worldwide, recently surpassing lung cancer in 2020. Globally, it is the number one cancer in both developed and underdeveloped countries and affects more than 2.3 million people, both men and women worldwide, despite its global abundance. Knowledge about the first steps in tumor initiation is important for early detection. However, the exact mechanisms of tumor initiation are still unknown. The median age of diagnostics is 62 to 63 years old, but more recent data also shows that breast cancer is the most common type of cancer among young women.
Ages 15-39 accounting for 30% of all cancers in this age group, understanding both the genetic and environmental makeup of the cancer will help drive better treatment for our patients. Mammographic density captured on film screen mammograms refers to the content and architectural structure of adipose, connective and epithelial tissues of the breast. In epidemiological studies, a high percentage of mammographic density confers up to a four to sixfold elevated risk of developing breast cancer. Mammographic density is to a large degree in inherited trait, although it is influenced by environmental factors as well. Levels of density are described using a reporting system called the Breast Imaging Reporting and Data System known as birads. The levels of density are a almost entirely fatty, which indicates that the breasts are almost entirely composed of fat. About one in 10 women have this result.
Fig-B (R) shows scattered areas of fibro-glandular density, which indicates that there are some scattered areas of density, but most of the breast tissue is non dense. About four in 10 women will have this result. Heterogeneously dense indicates that there are some areas of non dense tissue, but that most of the breast tissue is considered dense. About four in 10 women will have this result. Extremely dense indicates that nearly all of the breast tissue is considered dense. About one in 10 women have this result. This slide shows a box plot for mammographic density and breast cancer subtypes. Um, the percentage of mammographic density tumors in each of the subtypes are shown for basal HER2, LumA, LumB, and normal.
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TRANSCRIPT & VISUAL PRESENTATION
The first question people ask when there's a cancer diagnosis is "how do we treat it?" As you can see from the 109th annual meeting in Boston, we can find cancer of the breast and treat it with image guidance using ultrasound and MRI with focal therapies. Dr. Barens is now the current chair of research for the Tuck University in the Netherlands. So (enclosed) we see the cancer, it's dark on the ultrasound and it's vascular. We can mention the tumor vascularity and also with the the scan we see it's already metastasized. So we do everything staging with non-invasive imaging, non-bio, back to biopsy. Where do you biopsy? (Enclosed) is the mass. You put it here and get a good biopsy report or do you put it here and said that you, you got dead cells? Repeat the biopsy. So there are false positives, not only with biopsies and false negatives with biopsies, there are false positives.
PET scans, (enclosed) is a silicone in a lymph node from cosmetic surgery. (Enclosed is) a PET scan which showed three nodes but only one was positive with a 36% vessel density. This is the measure we use. This is the quantitative marker that shows how aggressive it is because when the 36 goes down to 2%, treatment is working. When it goes up to 55%, treatment is failing, start a different treatment. (Enclosed is) an advanced digital PET CT showing what are the nodes. So we have lots of tools in our armamentarium to find and to treat cancers. (Enclosed is) a metastatic rib focus and again, we can see that the tumor is high grade because there's a high vessel density and this is used to follow treatment. False positives. A rib fracture will show up on a PET CT scan as a cancer focus or a rib metastasis. This was thought to be benign keloid in this post-op area and the mass by the sterner is actually pitting and eroding into the sternal bone, which you can see on the sixth centimeter tumor that's just beneath the skin and it's breaking into the sternal bone at two points.
Another lump, just the fatty tumor is the skin. That black line is simply the epidermis. So we have fantastic imaging. This mass is a granuloma from the suture and this is a fat necrosis with calcification, both benign. (Enclosed is) a axillary suture fibrosis. This was missed by all imaging and the patient kept pointing on, this is where I heard. So we put the prob bon and this is a suture fibrosis. (Enclosed is) a good case. You (will) see the skin is abnormal. We did a thermogram, we showed it's highly positive. So the red is is tumor vascularity, same patient. This is the mass. Notice how the mass is eating into the bottom of the epidermis. So it changes it from a stage one to a stage two because it's spread to the skin.
Now this looks like a dense breast, but it isn't. This looks like a not dense breast, but it is because (enclosed is) the cancer. It's a huge six millimeter cancer on the outside of the breast and what does it look like with ultrasound? So is this a rash, a red breast? No, this is the epidermis again, and this is a five millimeter to six millimeter epidermal lymphatic cancer. Rare it's an inflammatory breast cancer rare, but this is the best way to find it. And sometimes the only way, again is an inflammatory cancer or a allergic reaction. Again, sonogram is 5.2 millimeters depth, which should be 1.2 with the allergen. And since the nipple and skin can be involved, we also use optical systems that see the microanatomy and see if the tumor has invaded the nipple tissue or the skin. How do we find that? Well, with looking at the skin, this is a intradermal white area.
It's a plaque, and the 4D scan shows multiple plaques. So this is uranium toxin, which is in the skin. You can find it and measure it. Let me leave you with the world conference showing that angiogenesis and vascularity are the key to finding cancer progression and seeing if the validated anti-cancer treatment works. (Enclosed is) something common. This is a reactive lymph node from from COVID. This is a metastatic lymph node. Notice the difference tumor vessels on the outside penetrating, and this is benign inflammatory vessels. (Enclosed is) a mass under the arm and a patient receiving biologics. So they thought it was a fatty tumor. No, it's a lymphoma. And notice this is connected with a vascular pedicle to the big subclavian artery. So you put a needle in (enclosed is) and it bleeds. This was courtesy from one of the heads of the ultrasound society in Europe. Notice the right side. This white line and blue line show that it's it's soft tissue area and on the left side the white line is thicker, thicker and now it's red, which shows that it's inflammatory tissue. So this is a case of benign fasciitis, which was treated with steroids instead of a metastatic tumor. So basically this is what the world is doing and we can be doing it by adopting all the new imaging technologies.
TRANSCRIPT & VISUAL PRESENTATION
I'm Dr. Roberta Kline. I'll be discussing the role of epigenetics. Gene expression is the final result of many systems interacting with each other. Now while alterations in DNA such as genetic SNPs and mutations are the best study, epigenetic changes are emerging as very important regulators, and both of these interact with the exosome or the sum total of all environmental exposures over a person's lifetime. These interactions are multi-directional. The exosome influences epigenetic changes. DNA affects how we respond to the exosome as well as our ability to create these epigenetic changes. And then the resulting genetic expression provides yet another feedback loop to influence all of these systems. Epigenetics literally means above the genome and it's our body's way of adapting to environmental cues without changing the actual DNA code. There are three main ways in which this occurs. There's methylation of DNA modification of histones, and then non-coding RNA.
Depending on the type, these can activate or inhibit gene expression. We'll now focus specifically on DNA methylation. DNA. Methylation occurs at very specific sites in the DNA sequence involving cytosine and guanine nucleotides. While we tend to see global trends in changes to methylation across the genome as we age, it's felt to be abnormal patterns associated with specific sites on specific genes that lead to increased risk of cancer. These patterns are most commonly seen as hypermethylation of sites that result in turning off tumor suppressor genes and hypomethylation of sites that result in turning on oncogenes. Unlike DNA epigenetic changes are dynamic and DNA methylation, therefore is a reversible process. Specific enzymes called DNA methyl transferases are responsible for both attaching and removing the methyl group that comes from snail methionine as the universal methyl donor. Snail methionine is the connection between epigenetics and folate and the broader pathways of trans methylation and transation, along with their genes.
As we learn more about what causes higher breast density at the molecular and genetic levels, the role of epigenetics is emerging. In a recent epigenomic wide association study, hypermethylation of a number of regions called differentially methylated regions or DMR were found to be associated with higher breast density. These same regions overlap significantly with ones that have been associated with breast cancer, highlighting potential common pathways and mechanisms mediated through epigenetics. In addition to specific functional pathways involving oncogenes and tumor suppressor genes, there is evidence that epigenetic alterations in genes linked to the microenvironment itself also play a role in breast density and breast cancer. These epigenetic changes are in genes across multiple biological systems that are involved in the maintenance of the microenvironment and disruptions are associated with increased breast density as well as with the development of progression of breast cancer. Soon we'll be able to go beyond hormone receptor and HER two status to provide even more personalized strategies using epigenetics.
Resistance to chemotherapy is also a huge concern and a hot topic of research that is now pointing to epigenetics as a potential solution. There are multiple clinical trials ongoing in various phases looking at intervention with some of these epigenetic agents. Most of these are focusing on two main enzymes, the methyl transferases, and another key epigenetic mechanism that involves enzymes called histone diacetyl asis. Nutritional epigenetics offers application of nutritional strategies including the use of specific phytonutrients that can modulate these epigenetic mechanism. Here on the left, you can see a list of various phytochemicals and how they interact with different epigenetic mechanisms or cancers including breast cancer. And on the right you can see this pictorially in terms of how they're acting with the epigene. Now these strategies are ones we can use today as part of a comprehensive personalized approach for the prevention and the treatment of elevated breast density and breast cancer.
Thank you.
TRANSCRIPT & VISUAL PRESENTATION
By: Dr. Noelle Cutter (Part 2)
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Molecular profiling of gene expression of breast cancers has demonstrated that tumors are remarkably heterogeneous. More recently, molecular analysis of the microenvironment has demonstrated similar heterogeneity, but the epidemiological clinical and pathological correlations of this variation are not well studied. Recent advances of breast cancers and the surrounding and microenvironment have revealed important stromal and epithelial interaction and have led to the speculation that the microenvironment may actually be dominant over tumor biology, especially early in the stage of progression when invasive cancer cells are still forming.
Clinical questions we ask in the lab are centered around identification of genetic signatures in that microenvironment of patients with dense breasts. If we can identify molecular changes, we can identify targets that are druggable using publicly available data to identify subsets of genes that are differentially expressed in breast cancer and correlated with high mammographic density. To identify important biological pathways is one of the methods that we've been utilizing in our labs. Um, using a bioinformatic parameter, we wanted to establish an in vitro, invitro 3D cell culture analysis that will accurately represent mammographic density. We are also interested in studying how gene suppression can be mimicked through the epigenetic mechanism of methylation. The methylation should induce suppression of transcription factors and that can be modeled within our in vitro system using a CRISPR Cas nine system. Once introduced into the cells growth characteristics and functional features can be studied. We propose that the analysis of these candidate genes that are identified in our study will help physicians make better clinical decisions when treating their patients.
We used a subset of genes quantified by the cancer genome atlas, which represent copy number variation where the CNV is low and downregulation of gene expression is seen, or we can look at copy number variations and gene upregulation for our functional genomic study. We next correlated this data to methylation changes across the genome using a platform known as MoMA, which is a methylation detection array. The image presented represents a sample representation of the heat met, which included 749 differentially methylated probes showing the segregation of mammographically dense and non mammographically dense patients with breast cancer. Since this analysis is based on the comparison of the two co cohorts of mammographically dense and non mammographically dense, there are regions that are found more frequently methylated in mammographically dense or non mammographically dense, breaking down to approximately 60% methylated in mammographically dense and 40% methylated in non mammographically dense tumors. For these regions that differentiate between the mammographically dense and non mammographically dense, we next determine which methylation event potentially represses transcription.
DNA methylation is an important epigenetic modification that defines the properties of cells. Genome-wide hypomethylation as well as hypermethylation of CPG islands is associated with tumor suppressor genes and developmental regulators and are characteristics of cancer cells changes in DNA methylation patterns associated with carcinogenesis progression gradually with the cell proliferation. CPG island methylation primarily targets promoters characterized by low gene expression. DNA methylation is a chemical modification that defines cell type and line lineage through the control of gene expression and genome stability. Disruption to the patterns of DNA methylation control mechanisms that are contributed to a bunch of diseases, especially cancer. Cancer cells are characterized by a barrant methylation such as genome-wide hypomethylation, and site-specific CPG hypermethylation, mainly targeting those CPG islands in gene expression regulatory elements. In particular, the early findings that a variety of tumor suppressor genes are target of DNA Hypermethylation in cancer has led to the proposal of a model in which a Barr DNA methylation promotes cellular oncogenesis through tumor suppressor gene silencing.
The cancer genome atlas, which I referred to earlier in this talk, is a comprehensive and coordinated effort to accelerate our understanding of the molecular basis of cancer through the application of genome analysis technologies, including large scale genome sequencing. The collaborative effort of the cancer Genome Atlas program advances personalized medicine.
When we analyze our subset of genes identified by the cancer genome atlas and shared publicly that are differentially expressed in patients with breast cancer and high mammographic density, we then uploaded our methylation gene list to a bioinformatic platform known as David to help identify any enriched themes in um, biological functionality for the subset of our genes. The David platform is the database for annotation visualization and integrated discovery and provides a comprehensive set of functional annotation tools for investigators to understand the biological meaning behind some of the large gene lists, such as the one that we isolated for our mammographically dense and our non mammographically dense patients. The star on the slide indicates pathways that are currently under validation in our lab and the hashtag indicates publications that have already been accepted for our gene analysis. That list.
This slide (R) shows a subset of genes that are both downregulated transcription repressed and methylated from our large subset of genes, which included the 749 probes found in the MoMA data. Also highlighted on this slide are two of the genes which we found upregulated in our dataset that have gone on for further functional analysis in our lab analysis of a selection of genes up upregulated in breast cancer and associated with epithelial to mesenchymal transition or EMT was performed utilizing the cancer genome atlas set and the database for functional annotation visualization and integrated discovery. Using the DAVID platform for pathway analysis, our results indicated an upregulation of two genes, tumor necrosis factor alpha in mammographically dense and non mammographically dense breast cancer cells, and the upregulation of zinc finger ebox binding protein one or ZEB one only. In the mammographically dense cells, TNF alpha is a pro-inflammatory cytokine and ZEB one is a transcription factor which directly affects the chromatin. We know that ZEB one regulates e deering expression and is a major cell to Sian protein and known to be a tumor suppressor protein in some cancers. Downregulation of eide and primes the detachment of cancer cells increases migration and invasion as well as metsis. Because of the crucial role of ZB one in this derrin regulation, ZEB one is considered a key regulated of EMT and tumor metastasis.
Our studies have shown that gene expression upregulation of these two key genes, ZB one and TNF Alpha, also induced functional changes in our cell lines, which include cellular proliferation and invasion. Being that TNF alpha and ZB one promote cell displacement and invasive myth through that EMT pathway, we hear and propose that these genes employ a pathogenic mechanism to render mammographically dense cells to metastasize. EMT is an overly complex but also reversible event. Therefore, further investigation into these genes in the an inhibition of either TNF alpha or Zev one might be an effective strategy for personalized medicine and cancer therapy.
Breast cancer tumors consist not only of tumor cell and cancer stem cells, but contain other cells within the tumor promoting functions such as cancer associated fibroblasts, normal fibroblasts myofibroblasts, mesenchymal stem cells, tumor associated adipocytes, endothelial cells, and various immune cells. These could include macrophages, neutrophils, natural killer cells, and regulatory T cells. We are currently in the process of further functional validation of our subset of genes and utilization of our three D cell culture model to model dense breast tissue in women Translational relevance.
Mammographic density is the strongest risk factor for nonfamilial breast cancer among women apart from older age, but its mechanistic underpinnings are poorly understood. We hypothesize that mammographic density would be associated with different subtypes of breast cancer based on their defining molecular pathways. Some of those pathways have be, have been delineated in our functional analysis study. Our results show that these well-defined molecular subtypes of normal tissue are strongly associated with both mammographic density and breast tissue composition, establishing novel molecular correlations of mammographic density to our gene expression analysis.
Many of the pathways enriched in patients such as epithelial to mesenchymal transition with higher mammographic density are certainly targetable raising the possibility of developing prevention strategies for mitigating density associated breast cancer risk. Future work in the lab will include clinical analysis with the Bard Cancer Center to validate some of our in vitro findings through both our expression array data, our gene subset, as well as our three dimensional cell culture analysis. We will continue to validate the three D cell culture model as well as confirm some of the genetic changes and epigenetic changes that we have already seen in our subset of gene. We plan to do further functional analysis to test the validated candidates and then provide a platform where we can undergo a personalized medicine approach using targeted therapeutic approaches.
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