Genetic Markers of Disease Lecture Notes PDF

2/7/2025 Genetic Markers of Disease COM5081 Fundamentals of Pathology Lecture 9 Kelsey Reindel, D.O. Assistant Professor, Family Medicine & OPP Dr. Kiran Patel College of Osteopathic Medicine, Nova Southeastern University. 1 Genes Gene: A...

2/7/2025 Genetic Markers of Disease COM5081 Fundamentals of Pathology Lecture 9 Kelsey Reindel, D.O. Assistant Professor, Family Medicine & OPP Dr. Kiran Patel College of Osteopathic Medicine, Nova Southeastern University. 1 Genes Gene: A segment of DNA that provides the cell with instructions for making a specific protein, which then carries out a particular function in your body. Nearly all humans have the same genes arranged in roughly the same order and more than 99.9% of your DNA sequence is identical to any other human. How are we different? On average, a human gene will have 1-3 letters (nucleotides) that differ from person to person. These differences are enough to change the shape and function of a protein, how much protein is made, when it's made, or where it's made. They affect the color of your eyes, hair, and skin, etc. More importantly, variations in your genome also influence your risk of developing diseases and your responses to medications. 2 The Human Genome Project The Human Genome Project (HGP) is one of the greatest scientific feats in history. The project was a voyage of biological discovery led by an international group of researchers looking to comprehensively study all of the DNA (known as a genome) of a select set of organisms. Launched in October 1990. April 2003– generating the first sequence of the human genome – provided fundamental information about the human blueprint, which has since accelerated the study of human biology and improved the practice of medicine. 3 1  2/7/2025 HGP Milestones June 2000 International Human Genome Sequencing Consortium announced that it had produced a draft human genome sequence that accounted for 90% of the human genome. The draft sequence contained more than 150,000 areas where the DNA sequence was unknown because it could not be determined accurately (known as gaps). April 2003 The consortium announced that it had generated an essentially complete human genome sequence, which was significantly improved from the draft sequence. Specifically, it accounted for 92% of the human genome and less than 400 gaps; it was also more accurate. March 2022 The Telomere-to-Telomere (T2T) consortium announced that it had filled in the remaining gaps and produced the first truly complete human genome sequence. 4 Genetic Variants Changes in the DNA sequence are called genetic variants. Genetic variants can occur in both coding and non-coding regions. Most of the time genetic variants have no effect at all. But, sometimes, the effect is harmful: just one letter missing or changed may result in a damaged protein, extra protein, or no protein at all, with serious consequences for our health. (pathogenic variant) SNP/SNV( single-nucleotide polymorphisms/single-nucleotide variations) Indels (insertions and deletions) Copy Number Variations Translocations and Inversions 5 Genetic Disorders Genetic Disorder: A disease caused in whole or in part by a change in the DNA sequence away from the normal sequence. Some diseases are caused by mutations that are inherited from the parents or arise spontaneously during cell division and are present in an individual at birth. Other diseases are caused by acquired mutations in a gene or group of genes that occur during a person's life. 1. Chromosomal: This type of genetic disorder affects the structures that hold your genes. With these conditions, people are missing or have duplicated chromosome material. (Down Syndrome, Turner Syndrome, Klinefleter Syndrome, Trisomy 18, Trisomy 13, etc.) 2. Complex (Multifactorial): These disorders stem from mutations in multiple genes (multifactorial inheritance disorder), by a combination of gene mutations and environmental factors, or by damage to chromosomes (Cancers, Diabetes, CAD, Autism Spectrum Disorder, Alzheimer’s Disease, etc.) 3. Single-Gene (Monogenic): This group of genetic disorders occurs from a single gene mutation (CF, DMD, Congenital Deafness, Hemochromatosis, NF1, Sickle Cell Disease, Tay-Sachs Disease, etc.) The passing of genetic variants from one generation to the next helps to explain why many diseases run in families. 6 2  2/7/2025 Genetic Markers Genetic markers: are variants in the DNA code at a known physical location on a chromosome that, alone or in combination, are associated with a specific disease phenotype. Markers whose presence confers a high level of probability of disease (a “high predictive value”) would be most useful as diagnostic tools or as predictors of prognosis or response to therapy. Single nucleotide polymorphisms (SNPs, or variants at a single DNA base pair) have been the focus as potential genetic markers. They have the advantage of a high frequency in the human genome (1 occurs every 1000 nucleotides, on average) and are relatively easy to genotype using current technologies. 7 https://youtu.be/HwEAjZWPw2k Genetic Testing Genetic tests have been developed for thousands of diseases. Most tests look at single genes and are used to diagnose rare genetic disorders However, a growing number of tests are being developed to look at multiple genes that may increase or decrease a person’s risk of common diseases, such as cancer or diabetes. Such tests and other applications of genomic technologies have the potential to help prevent common diseases and improve the health of individuals and populations. 8 9 3  2/7/2025 10 Gene Sequencing A large investment has been made in improving DNA sequencing technologies, to make them cheaper, faster, and more accurate. The following terms are used to distinguish sequencing methodologies: 1.Sanger Sequencing/1st Generation 2. Next Generation Sequencing (NGS)/2nd Generation 3 main types 3. Third Generation Sequencing*** 11 Sanger NGS 12 4  2/7/2025 13 Sanger Sequencing Dr. Frederick Sanger developed Sanger sequencing over 40 years ago. This process sequences short DNA regions of interest (~300-1000bp) This method is the “gold standard” of sequencing methods because of its high accuracy. Conducting larger sequencing projects with Sanger sequencing quickly becomes expensive and time-consuming. Designing primers for entire chromosomes is not a viable option for high-throughput, large-scale sequencing projects. 14 Single Gene Sequencing- Sanger For Example: Cystic Fibrosis is a disease with a mutation in the CFTR Gene (This gene has a certain sequence of nucleotides: ATCG…) This sequence will give rise to mRNA which will give rise to an amino acid/protein There is a change in one base within the sequence that gives rise to a change in the protein ultimately causing the disease If we can see all of the nucleotides that code for a gene then we can identify the change in one of the base nucleotides causing the mutation/disease This single gene sequencing is great for monogenic diseases (ie, caused by defects in only one gene). However, for more common diseases, it has been more difficult to identify genetic markers, because most common diseases are polygenic and often triggered by an interaction of genetic, environmental, and physiological factors, making it difficult for researchers to narrow their focus to a single gene. In these cases, a “genomic” approach that examines the entire genome may be valuable. 15 5  2/7/2025 Next Generation Sequencing/2nd Generation The first next-generation sequencing (NGS) technology was released in 2000, by companies who would later become part of Illumina. These technologies made it possible to sequence whole genomes in much less time. For context, Sanger sequencing can cover between 300 and 1,000 nucleotides at one time. The NovaSeq 6000 S4 flow cell is capable of sequencing 48 human genomes (144 billion base pairs) in about two days — that is the power of NGS. NGS can be used to sequence every nucleotide in an individual's DNA (ie, the whole genome), or limited to smaller portions of the genome such as the exome or a preselected subset of genes Exome Sequencing Whole Genome Sequencing Multiple Gene Sequencing Panel NGS technologies have expanded our knowledge of cellular functions and disease mechanisms. Whole exome sequencing, transcriptomics, and proteomics tools give researchers insights into all levels of biology. These NGS tools have opened countless doors in disease research, helping us better understand things like inflammation, drug resistance, and oncology 16 NGS-How it works Extract DNA from any source that provides DNA (Saliva, Buccal, Hair etc.) DNA extracted from leukocytes in whole blood is a sterile source of DNA used for most clinical testing. Take the single-stranded DNA and cut it into 200-250 base pair sections Then you will use PCR to amplify each segment We know the “Normal Human Genome Library” from chromosomes 1-22, X/Y NGS “matches” the amplified bp segments to the Library within the computer and data is interpreted 17 Exome Sequencing The exome contains the portions of genes that encode proteins; it represents only 1.5 to 2.0 percent of the genome. The remaining (non-exomic) DNA consists of introns and regulatory regions that control other aspects of gene function such as splicing and gene expression levels. Exome sequencing is a reasonable approach for some clinical situations because over 85 percent of known disease-causing mutations are found in exons. This approach substantially reduces cost and data storage requirements compared with whole genome sequencing. The main disadvantage of exome sequencing over whole genome sequencing is that exome sequencing could potentially miss a pathogenic variant(s) in a non-coding region of the genome. Thus, whole genome sequencing may be used in selected cases if initial exome sequencing is not diagnostic. 18 6  2/7/2025 Whole Genome Sequencing Whole genome sequencing is costlier than more limited sequencing because the whole genome is equivalent to approximately 3.3 x 109 bases (3.3 gigabases [Gb]). Whole genome sequencing may become preferable to exome sequencing as cost decreases and more information about the role of non-coding DNA(Introns) in human disease becomes available. 19 Targeted Gene Panels Gene panels provide sequence data for a limited subset of genes (typically 10 to 200 genes). Targeted gene panels are used in settings in which it would be appropriate to sequence many genes to make a diagnosis (a disorder for which the number of candidate genes is too large for traditional Sanger sequencing). As an example, certain diseases’ potential etiologies include pathogenic variants in over 60 genes, and screening for all of these individually would be impractical using traditional sequencing methods. Targeted gene panels may be preferable to exome and genome sequencing due to the considerable cost advantage, the lower likelihood of identifying variants of unknown significance that are unrelated to the disease being evaluated The utility of targeted gene panels over Sanger sequencing continues to increase as costs decline and as these gene panels offer a wide enough net of genes to assay for many conditions. The number of available panels also continues to increase, with validated panels available for hereditary cardiomyopathies, inherited cancer syndromes, lung diseases, ciliopathies, and other disorders in which molecular diagnosis is better facilitated by sequencing multiple genes known to be causative in the majority of cases. 20 Accuracy Accuracy for targeted NGS gene panels is higher since sequencing a smaller region of the genome allows for a greater degree of probe-template overlap compared to exome and genome sequencing. Sanger sequencing remains the "gold standard" for diagnosis based on gene sequencing, with >99.99 percent accuracy reported for most genes sequenced. Clinical laboratories generally perform Sanger sequencing to confirm any variant reported back to the ordering clinician as pathogenic, because of the greater accuracy of Sanger sequencing. **However, given the continued improvement of NGS-based technology, the necessity of performing secondary validation with Sanger sequencing is being challenged 21 7  2/7/2025 Genetic Markers in Cancer Screening and Management Targeted gene panels have shown expanded usefulness across many cancer types, especially those for which more than one genetic variant may be responsible. Multi-gene panels for certain inherited cancer syndromes based on National Comprehensive Cancer Network (NCCN) recommendations are becoming increasingly popular options for certain patients as the field moves away from single- gene testing to the panel approach. This approach may be more efficient and cost- effective, given the decreasing cost associated with NGS technology and increasing indications for genetic testing 22 Breast Cancer and Genetic Testing Breast cancer is the second most common cancer in women after skin cancer. Each year, approximately 200,000 women in the United States are diagnosed with breast cancer, and one in nine American women will develop breast cancer in their lifetime. The HCP will assess risk factors and determine whether a patient is at average or increased risk for developing breast cancer 23 If your residual lifetime risk is 20% or more, it may be recommended to have a genetic evaluation and should be referred to a genetic counselor. 24 8  2/7/2025 Breast Cancer and Genetic Testing The most common implicated pathogenic variants in these types of patients occur in the BRCA1/2 genes, which are inherited in an autosomal-dominant fashion. It is recommended to use a cancer gene panel that includes BRCA1 and BRCA2 if there is a personal or family history of prostate and/or pancreatic cancer, even in the absence of breast or ovarian cancer. Other genes for which pathogenic variants may be associated with increased breast cancer risk include ATM, CDH1, CHEK2, NF1, PALB2, and TP53. For most patients (irrespective of age) meeting National Comprehensive Cancer Network (NCCN) criteria for testing, next-generation multigene panel testing is offered. Although pathogenic variants in breast cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2 [BRCA1/2]) are the most common implicated in women with classic signs of hereditary breast/ovarian cancer, approximately 4-7% have a pathogenic variant in another gene with probable breast and ovarian cancer associations. checkpoint kinase 2 (CHEK2) partner and localizer of BRCA2 (PALB2) ataxia-telangiectasia mutated (ATM) 25 Genetic Testing and Guide to Treatment in Breast Cancer For those diagnosed with breast cancer, some inherited gene mutations put you at a high risk of getting breast cancer in the opposite breast or delineate a more aggressive type of cancer A healthcare provider may recommend you consider a mastectomy instead of a lumpectomy. Women with some inherited gene mutations may also consider a risk-reducing contralateral (opposite breast) prophylactic mastectomy. Women with a high-risk mutation in one of these genes may consider risk-reducing contralateral mastectomy to lower their risk of breast cancer in the opposite breast: BRCA1 BRCA2 CDH1 PALB2 PTEN STK11 TP53healthcare 26 Genetic Testing and Guide to Treatment in Breast Cancer Genetic testing is even leading to more effective therapeutics that reduce the chance of relapse or drug resistance. 1. PARP (Poly ADP-ribose Polymerase) Inhibitors and BRCA1 and BRCA2 inherited gene mutations in breast cancer 1. PARP is an enzyme that helps repair damaged DNA. 2. PARP inhibitors ultimately keep cancer cells from repairing damaged tumor DNA 3. BRCA1/2-related breast cancers are more dependent on PARP in repairing tumor DNA after chemotherapy or radiation. Treating BRCA1/2-related breast cancers with a PARP inhibitor makes it harder for the breast cancer to repair itself, leading to tumor cell death 4. PARP inhibitors are used for the treatment of HER2-negative early breast cancer at high risk of recurrence in people who have BRCA1 or BRCA2 (BRCA1/2) inherited gene mutations and have been treated with chemotherapy and/or radiation. 5. Olaparib (Lynparza), Talazoparib (Talzenna), niraparib (Zejula), rucaparib (Rubraca) The phase III OlympiA trial found that adjuvant treatment with olaparib, a PARP inhibitor, extended disease-free survival in patients with inherited BRCA1/2 pathogenic variants who had high-risk, early- stage, human epidermal growth factor receptor 2 (HER2)-negative early breast cancer. Thus, breast cancer patients who may benefit from this treatment are recommended to undergo germline genetic testing Study findings have shown people with a BRCA1/2 gene mutation who had HER2-negative breast cancer at high risk of recurrence and got olaparib had a lower risk of breast cancer recurrence and better survival than those who didn’t get olaparib. 27 9  2/7/2025 Genetic Testing and Guide to Treatment in Breast Cancer Genetic testing is even leading to more effective therapeutics that reduce the chance of relapse or drug resistance. 1. HER2- Human Epidermal Growth Factor Receptor 2 1. Membrane tyrosine kinase receptor that provides the cell with potent proliferative and anti-apoptosis signals 2. when activated or overexpressed, breast cells can multiply too quickly causing a tumor. 3. The purpose of HER2 testing is to determine whether cancer cells have too many copies of the HER2 gene or a higher-than-normal level of the HER2 protein. Cancers with higher-than-normal levels of HER2 are called “HER2-positive.” Cancers with normal HER2 levels are called “HER2-negative.” 4. Breast cancers that are positive for HER2- tend to be more aggressive and recur HER2-targeted treatments such as trastuzumab, pertuzumab, or lapatinib may be part of the treatment plan for patients with HER2-positive cancer. If the tumor is HER2-negative, HER2-targeted therapies are not effective. 28 Genetic Testing and Guide to Treatment in Breast Cancer Genetic testing is even leading to more effective therapeutics that reduce the chance of relapse or drug resistance. 1. HR+- Hormone Receptor-Positive Breast Cancer 1. Proteins found on breast cells that pick-up estrogen or progesterone signals that promote cell growth, including cancer cell growth 2. Breast cancer cells that have receptors for either hormone are considered HR+ (ER, PR, or both) 3. If a cancer cell is HR+, it can receive signals from estrogen/progesterone to grow Knowing whether the tumor needs estrogen and/or progesterone to grow makes it easier to treat the cancer and a more targeted approach. Hormone Therapy: used to stop estrogen hormones from fueling breast cancer cells to grow that are HR+. SERMs- Selective Estrogen Receptor Modulators- block estrogen from binding to cancer cells. They have anti-estrogen effects on breast cells but do not affect other tissues like the uterus and bones (Tamoxifen) SERDs- Selective Estrogen Receptor Degraders- bind to the receptors more tightly and break them down. They have anti-estrogen effects throughout the body and therefore only given to women post-menopausal (Fulvestrant, Elacestrant) AIs- Aromatase Inhibitors- Drugs that stop most estrogen production in the body (Letrozole, Anastrozole, Exemestane) 29 Oncotype DX Oncotype DX tests a sample of the tumor (removed during a biopsy or surgery) for a group of 21 genes. These genes are in the tumor cells, not in the normal (non-cancer) cells in a person’s body. Oncotype DX is the most common tumor profiling test used in the U.S. and the only one used in breast cancer staging. Predicts chance of future metastasis Predicts the likelihood that treatment with chemotherapy in addition to hormone therapy will be helpful Informs treatment decisions and improves confidence in treatment decisions 30 10  2/7/2025 Ovarian Cancer and Genetic Testing Ovarian cancer most often occurs for unknown reasons however 1 in 6 is due to hereditary ovarian cancer. Hereditary ovarian cancer is most often caused by mutations in BRCA1 or BRCA2. Ovarian Cancer associated with BRCA Mutations or with Lynch Syndrome usually starts at a younger age BRCA Mutation in ovarian cancer is the most important biomarker in planning treatment as well 31 Prostate Cancer and Genetic Testing Prostate cancer is the most common cancer in males worldwide, with over 1.2 million cases and 358,000 deaths in 2018 according to data from the GLOBOCAN database. The most important known risk factors for prostate cancer are AGE, ethnicity, and inherited genetic variants/Family History. Individuals not yet diagnosed with prostate cancer, a family cancer history (in first- and second- degree relatives), including the type(s) of cancer, and age at diagnosis may help identify individuals who may carry genetic factors that increase the risk of developing prostate cancer and potentially other cancers. If the family history suggests this possibility, providers should discuss referral to genetic counseling and germline genetic testing. 32 Genetic Markers is Prostate Cancer Males with germline mutations in BRCA2, in particular, are at increased risk of prostate cancer and have more aggressive disease features. Studies show for BRCA2 mutation carriers, the relative risk of prostate cancer is estimated to be increased 2.2- to 8.6-fold compared with noncarriers Guidelines from the NCCN recommend for anyone with a personal or family cancer history of high-risk germline pathogenic variant; prostate cancer screening for BRCA2 mutation carriers starting at age 40 years, given that these individuals may have an increased risk of early and potentially lethal prostate cancers before age 65 years, and that screening be considered at annual rather than every other year intervals The Philadelphia Prostate Cancer Consensus Conference 2019 recommended that BRCA2 mutation status be factored into prostate cancer screening discussions, with a baseline PSA at age 40 years or 10 years prior to the youngest prostate cancer diagnosed in the family 33 11  2/7/2025 Genetic Testing and Guide to Treatment in Prostate Cancer Genetic testing is even leading to more effective therapeutics that reduce the chance of relapse or drug resistance. Finally, BRCA2 mutations might inform decision-making about active surveillance 1. The identification of a germline mutation in BRCA2 or other DNA repair genes may have implications for treatment in males with metastatic prostate cancer 1. Patients with metastatic castration-resistant prostate cancer (CRPC) carrying BRCA2, BRCA1, or ataxia telangiectasia mutated (ATM) mutations have similar progression-free and overall survival with both abiraterone and enzalutamide compared with those without such mutations 34 Colorectal Cancer The risk factors for colorectal cancer (CRC) are both environmental and inherited. The mode of presentation of CRC follows one of three patterns that are reflective of these differing risk factors: 1. Sporadic 2. Familial 3. Inherited Sporadic disease, in which there is no family history, accounts for approximately 70 percent of all CRCs. It is most common over the age of 50, and dietary and environmental factors have been etiologically implicated "familial" CRC, which accounts for up to 25 percent of cases. Affected patients have a family history of CRC, but the pattern is not consistent with one of the inherited syndromes described below. Fewer than 10 percent of patients have a true inherited predisposition to CRC, and these cases are subdivided according to whether or not colonic polyps are a major disease manifestation. 1. The diseases with polyposis include familial adenomatous polyposis (FAP), MUTYH-associated polyposis (MAP), and the hamartomatous polyposis syndromes (eg, Peutz-Jeghers, juvenile polyposis) 2. Those without polyposis are referred to as hereditary nonpolyposis CRC (HNPCC; Lynch syndrome). 3. These conditions are all associated with a high risk of developing CRC. In many cases, the causative genetic mutation has been identified, and a test is available. 35 Biomarker Testing in CRC Lynch Syndrome is a type of inherited cancer. People with Lynch syndrome are more likely to get colorectal cancer and other cancers, and at a younger age (before 50) including ovarian, endometrial, stomach, and skin cancers. It is caused by inherited mutations of genes that fix damaged DNA called mismatch repair (MMR) genes. When the MMR system is not working, errors build up and cause the DNA to be unstable which is called microsatellite instability (MSI). MLH1, MSH2, MSH6, PMS2, and EPCAM NCCN recommends everyone diagnosed with colon cancer be tested for MMR gene mutations to determine who should be tested for Lynch Syndrome. If one is MMR Deficient or has High MSI- TESTED 36 12  2/7/2025 Biomarker Testing in CRC Among the oncogenes implicated in sporadic CRC are RAS, SRC, MYC, BRAF, and the human epidermal growth factor receptor 2 (HER2) Most important is RAS- mutated in approximately 40% of CRC cases The identification of RAS mutations in CRC is of potential clinical relevance for both screening and therapy: 1. Screening: Detection of RAS mutations, in combination with aberrantly methylated BMP3 and NDRG4 promoter areas, beta-actin, and a test for stool hemoglobin (FIT) are included in multi- target stool DNA testing for colon cancer (Cologuard). the sensitivity for CRC detection with cologuard was 92% compared with FIT alone at 74% 2. Treatment: Patients with RAS-mutated mCRC do not benefit from agents targeting the epidermal growth factor receptor (EGFR), as these activating mutations induce resistance to EGFR inhibitors via constitutive activation of the RAS pathway. 37 Updates in Genetic Testing and SUD Drug use and addiction represent a public health crisis with more than 46 million people in the U.S. >12 had at least one substance use disorder Substance use disorders are heritable and influenced by complex interactions among multiple genes AND environmental factors Environmental factors such as trauma, stress, etc can influence whether or not you use drugs/alcohol however research shows that a person’s genes can account for 40-60% of that individual’s predisposition to alcohol or drug addiction Scientists have recently identified genes commonly inherited across addiction disorders, regardless of the substance being used. 19 independent SNP’s significantly associated with general addiction risk were found People with lower levels of CHRNA2 gene may be for likely to develop cannabis use disorder The strongest gene signals consistent across various disorders mapped to areas in the genome known to control the regulation of dopamine signaling, suggesting regulation of signaling vs signaling itself is central to addiction risk The hope with genomic studies and substance use disorder is to illuminate factors that may protect or predispose a person to substance use disorders- knowledge that can be used to expand preventative services and also develop personalized interventions that are tailored to an individual’s unique biology. Psychiatrists can now use genetic testing to determine which prescription medications work best for treating each particular addiction. A variant in GRIK1 may influence a physician to prescribe Topomax instead vs Naltrexone in persons with a variant in OPRM1 to curb alcohol cravings Genetic Testing is NOT the holy grail for diagnosing and treating addiction. This is just used as a guide in certain circumstances but could also be the future…. A 38 The Future Polygenic Risk Scores: Can provide a measure of your disease risk due to your genes Combining polygenic risk scores with other factors that affect disease risk can give you a better idea of how likely you are to get a specific disease Studies are looking at how useful polygenic risk scores are in real-life clinical practice This information on how each gene change affects disease risk comes from population-level genetic studies like GWAS GWAS- genome-wide association studies: involves rapidly scanning markers across complete sets of DNA of many people to find genetic variations associated with a particular disease https://youtu.be/HwEAjZWPw2k 39 13  2/7/2025 DNA is not your Destiny. The way you live influences how your genome works. 40 References https://www-uptodate-com.ezproxylocal.library.nova.edu/contents/tests-for-screening-for-colorectal- cancer?sectionName=Multitarget%20stool%20DNA%20tests%20with%20fecal%20immunochemical%20testing&search=gentics%20and%20 colon%20cancer&topicRef=2485&anchor=H240481080&source=see_link#H6 https://www.broadinstitute.org/delivering-gene-based-therapies#top https://pubmed.ncbi.nlm.nih.gov/29236593/ https://pubmed.ncbi.nlm.nih.gov/34003218/ https://www.nccn.org/patients/guidelines/content/PDF/ovarian-patient.pdf https://www.nccn.org/patients/guidelines/content/PDF/colon-patient.pdf https://www.nccn.org/patients/guidelines/content/PDF/prostate-early-patient.pdf https://www.nccn.org/patients/guidelines/content/PDF/breastcancerscreening-patient.pdf https://globalgenes.org/toolkit/genetic-testing-is-this-my-path-to-a-diagnosis/ https://www.psomagen.com/blog/rise-of-third-generation-sequencing https://www.uptodate.com/contents/molecular-genetics-of-colorectal-cancer 41 14