Cancer Genetics and Genomics II PDF

Cancer Genetics and Genomics [2] Julie W. Hirschhorn, Ph.D., HCLD Office: (843) 792-1181 Email: [email protected] A. Inherited Cancers 1. Inherited Mutations 2. Inheritance pattern 3. Two-Hit Hypothesis 4. Clinical Features 5. Some Examples i. Retinoblastoma ii. Li-Fraumeni Syndrome iii. Familial Ad...

Cancer Genetics and Genomics [2] Julie W. Hirschhorn, Ph.D., HCLD Office: (843) 792-1181 Email: [email protected] A. Inherited Cancers 1. Inherited Mutations 2. Inheritance pattern 3. Two-Hit Hypothesis 4. Clinical Features 5. Some Examples i. Retinoblastoma ii. Li-Fraumeni Syndrome iii. Familial Adenomatous Polyposis iv. Von Hippel-Lindau Disease v. Lynch Syndrome vi. Inherited Breast Cancer vii. Peutz-Jeghers Syndrome B. Somatic Cancers 1. Role of the Environment 2. Role of Oncogenic Viruses C. Individualized/Precision Medicine in Oncology 1. Classic Cytotoxic Therapies 2. Molecular Profiling 3. Targeted therapies 4. Immunotherapies D. Laboratory Testing for Oncology 1. Familial Testing 2. Somatic Testing Recommended Reading: 1. Mark’s Basic Medical Biochemistry, 5th Ed. 2018. Chapter 18, Section: Viruses and Human Cancer. MUSC Library Online Link: https://meded.lwwhealthlibrary.com/book.aspx?bookid=2170 2. Henry’s Clinical Diagnosis and Management by Clinical Laboratory Methods, 23rd Ed, 2017, McPherson and Pincus: Chapter 71, pages 1393-1394. MUSC Library Online Link: https://www.clinicalkey.com/#!/browse/book/3-s2.0-C20130143425 3. Genetics in Medicine, 8th Ed, 2016, Thompson and Thompson: Chapter 15, Sections: Cancer in Families, Sporadic Cancer. MUSC Library Online Link: https://pascal- musc.primo.exlibrisgroup.com/permalink/01PASCAL_MUSC/6ubsj8/alma991000286805 105641 “The time is right to bring the full power of genomics to bear on the problem of cancer.” -Francis Collins (Director of the NIH) Page 1 of 18 OBJECTIVES 1. Describe the clinical features of familiar cancer syndromes. 2. Describe why familial cancer syndromes present as autosomal dominant traits in family pedigrees but have a recessive genetic mechanism. 3. Describe the two-hit hypothesis and how it contributes to early age of onset in hereditary cancers. 4. Specify molecular, contributing environmental factors and viruses underlying the mechanism of cancer development and progression. 5. Distinguish between targeted therapies and standard chemotherapy and radiation regimes. 6. List some methods of laboratory testing to identify familiar cancer mutations. 7. Differentiate between laboratory testing methods for somatic tumors and germline tumors. 8. Describe how oncogene addiction is related to the success of targeted therapies. Illustrations adapted from: • • • • Henry’s Clinical Diagnosis and Management by Clinical Laboratory Methods, 23rd Edition, © 2017, Elsevier Inc. Molecular Pathology in Clinical Practice, 2nd Edition, © 2016, Springer International Publishing Genetics in Medicine, 8th Edition, © 2016, Elsevier Inc. Mark’s Basic Medical Biochemistry, 5th Edition, © 2018, Wolters Kluwer Other References: Hanahan and Weinberg. The Hallmarks of Cancer. 2000. Cell. 100(7):57-70. Hanahan and Weinberg. Hallmarks of Cancer: The Next Generation. 2011. Cell. 144:646-674. Li et al. Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer. 2017. J Mol Diag. 19(1):4-23. Hoppe-Seyler et al, The HPV E6/E7 Oncogenes: Key factors for viral carcinogenesis and therapeutic targets. 2018. Trends in Microbiology. 26(2):158-168. Molecular Pathology in Clinical Practice, 2nd Ed, 2016, Leonard: Section II, pages 315-399. MUSC Library Online Link: https://link.springer.com/book/10.1007%2F978-3-319-19674-9 (this book is for deeper reference, you do not need to read or memorize) Page 2 of 18 NOTES A. INHERITED CANCERS 1. Inherited Mutations All cancers are genetic at the cellular level and caused by mutations. These variants may occur in proto-oncogenes or oncogenes that normally promote proliferation or in tumor suppressor genes which normally restrain cell growth. Hereditary cancer syndromes are disorders that may predispose individuals to developing certain cancers. Germline variants are inherited, present in the egg or sperm, and are the cause of family inherited cancers. Somatic variants occur in non-germline tissues and are not heritable. When related to cancer, variants generally lead to malignant transformation. The most commonly inherited variants that contribute to familial cancers syndromes are found in tumor suppressor genes. To date, researchers have associated mutations in specific genes with more than 50 hereditary cancer syndromes. De novo mutations are new variants that occur in a germ cell, so in these patients we would not expect to see a family history of hereditary cancer syndromes. De novo mutations are common in: Familial adenomatous polyposis ~ 30% of cases MEN 2B ~ 50% of cases Hereditary retinoblastoma ~ 50% of cases Neurofibromatosis, type 1 ~ 50% of cases Peutz-Jeghers syndrome ~20% of cases Von Hippel-Lindau disease ~20% of cases 2. Inheritance pattern Most familial cancer syndromes exhibit similar pedigrees and work by a similar mechanism (the two-hit hypothesis). In some cases, these inherited cancers will present as autosomal dominant disorders, but they are genetic recessive disorders that are quasi-autosomal dominant. For example, the RB1 gene is a tumor suppressor gene and encodes for the pRB protein. This protein is normal cells is involved in regulating cell growth and keeping the cell from dividing too quickly. Mutations in the RB1 gene can be passed through the germline in the hereditary cancer of retinoblastoma. Retinoblastoma is a rare type of eye cancer that usually develops in early childhood (before the age of 5). Therefore, the disease itself requires a recessive genotype, but often presents in families as a dominant disorder. To develop retinoblastoma, both copies of the RB1 gene must be mutated or lost in order for the cancer to form. How can this be explained? Page 3 of 18 3. Two-Hit Hypothesis Dr. Alfred George Knudson, Jr, a U.S. geneticist and pediatrician, presented his “two-hit” theory in 1971. Based on his clinical observations, the early age of onset of retinoblastoma, and the extent of disease in hereditary versus somatic cases, he developed the theory that tumor suppressor genes required a loss-of-function in both alleles to initiate tumor formation. Cancer, regardless of being hereditary or non-hereditary, requires two genetic events to initiate oncogenic transformation. The inherited mutation is the initiation event but is not sufficient to cause cancer. A second mutation is required to cause the cancer to form. Since the first mutation is already present at birth, the second event is likely to happen earlier in life. This mechanism explains the seemingly dominant inheritance pattern in families. In somatic tumors, the onset is often later in life because the two mutations in the same cell must be acquired and that takes time. Page 4 of 18 This pedigree is a stylized example of the two-hit hypothesis • In an autosomal dominant disease, if you inherit the abnormal gene from only one parent, you get the disease • But many of these are genetic recessive disorders that are quasiautosomal dominant • The loss of the second allele can happen by a variety of different mechanisms: 4. Clinical Features The most common features of inherited cancers are: • Early age of onset or diagnosis • Same cancer in two or more close relatives on the same side of the family • The disease is often multifocal • The disease appears bilaterally when in paired organs • The presence of multiple rare tumors in the same individual • Constellation of tumors consistent with a specific cancer syndrome. • Evidence of autosomal dominant transmission (e.g. multiple affected generations and the presence of congenital abnormalities or syndromeassociated benign lesions). There are several factors that can affect penetrance including modifier genes, Page 5 of 18 severity of the loss of the function of the gene or protein, response to DNA damage and ability to repair, carcinogens, and hormonal and reproductive factors that influence gene expression. 5. Some Examples of Familial Cancer Syndromes Hereditary Cancer Gene(s) Affected Somatic Cancer Retinoblastoma, small cell lung carcinomas, breast cancer Lung cancer, breast cancer, many other cancers Retinoblastoma Li-Fraumeni Syndrome (LFS) Familial adenomatous polyposis (FAP) Von HippelLindau Disease (VHLD) RB1 p110 cell cycle regulation TP53 p53 cell cycle regulation Colorectal cancer APC Clear cell renal cell carcinoma VHL Lynch Syndrome (LS or HNPCC) Colorectal cancer MLH1, MSH2, PMS2, MSH6, and MSH3 Familial breast and ovarian cancer Breast and ovarian cancer BRCA1 and BRCA2 Peutz-Jeghers Syndrome (PJS) Breast cancer, nonsmall cell lung carcinoma, ovarian cancer, melanoma, and pancreatic cancer STK11/LKB1 i. Protein and Possible Mechanism APC Regulation of proliferation and cell adhesion VHL Protein degradation and control of cell division MLH1, MSH2, PMS2, MSH6, and MSH3 Repair nucleotide mismatches between strands of DNA BRCA1 and BRCA2 Chromosome repair in response to double-strand DNA breaks Serine/threonine kinase 11 Regulation of proliferation, cell polarization, energy usage, and apoptosis regulation Retinoblastoma • Hereditary retinoblastoma presents as an autosomal dominant inheritance pattern • Most common primary intraocular malignancy of childhood associated with blindness and mortality (1 in 20,000 live births a year in the U.S.) • Retinoblastoma can also be non-heritable • Retinoblastoma is caused by the bi-allelic inactivation of the tumor suppressor gene, RB1. o The RB1 gene is a tumor suppressor gene and encodes the Rb1 protein, which functions as a nuclear phosphoprotein Page 6 of 18 involved in regulating the G1 to S phase transition. The Rb1 protein can bind and inhibit the function of the E2F family of transcription factors, which regulate several genes involved in S-phase entry. o The most common ways that RB1 is inactivated is through mutation, deletion, or by epigenetic mechanisms. ii. Li-Fraumeni Syndrome (LFS) • Rare, autosomal dominant inheritance pattern • Patients often present with multiple primary tumors at an early age (often childhood) o Childhood cancers include osteosarcoma, soft-tissue sarcoma, brain tumors, and adrenocortical carcinoma o Adult-onset cancer includes colorectal, breast, and others o The most described germline variants in LFS occurs in the TP53 gene:  Most of the mutations are found in the central DNAbinding domain, which can interfere with the ability of Tp53 to bind to its target DNA sequence o Approximately 2/3 of the tumors in LFS patients only carry a missense on one allele of the TP53 gene, leaving the other allele wild type. One explanation is that this may serve as a dominant negative effect and functionally inactivate the wild type TP53 copy. Another explanation is that these TP53 Page 7 of 18 variants may result in new p53 functions that contribute to tumor initiation and progression. Molecular Pathology in Clinical Practice, Figure 28.3 iii. Familial Adenomatous Polyposis (FAP) • FAP has an autosomal dominant inheritance pattern • FAP is estimated to account for approximately 1% of the colorectal cancers in the general population o The average age of diagnosis is 39 years old o Approximately 50% of mutation carriers develop adenomas by 14 years old • Mutations in the APC gene on chromosome band 5q21 have been Page 8 of 18 shown to cause FAP o Mutation types include single nucleotide substitutions, and small and large insertions and deletions • iv. The diagnostic criteria are dependent on the identification of hundreds of benign adenomatous polyps developing in the colon during the first two decades of life Von Hippel-Lindau Disease (VHLD) • VHLD is an autosomal dominant cancer predisposition syndrome • Point mutations in the VHL gene have been identified in virtually 100% of patients • VHLD can gives rise to hemangioblastomas of the brain and spine, retinal angiomas, clear cell renal cell carcinoma, pheochromocytoma, endolymphatic sac tumors, tumors of the epididymis or broad ligament, and pancreatic tumors or cysts. o Onset of disease is usually between 40 and 50 years of age o Penetrance for the disease is virtually 100% by the age of 65 Page 9 of 18 v. Lynch Syndrome, also known as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) • Autosomal dominant inheritance pattern • This hereditary cancer syndrome is associated with increased risk for colorectal, uterine, and other cancers o The overall risk of developing colorectal cancer by age 70 in Lynch carriers has been reported to be as high as 82%, with risk of uterine cancer in women as high as 60% o There is a tendency toward a right-sided proximal location in the colon for these tumors The genes involved in this hereditary cancer syndrome belong to • Page 10 of 18 the DNA mismatch repair family o 90% of Lynch cases presenting with mutations in the MLH1 and MSH2 genes o Additional MMR associated with Lynch syndrome include PMS2, MSH6, and MSH3 Molecular Pathology in Clinical Practice, Table 24-1 vi. Inherited Breast Cancer • Inherited mutations explain 10% of all breast cancer cases, majority of these mutations are in the BRCA1 and BRCA2 genes • Hereditary breast-ovarian cancer syndrome (HBOC) diagnosis is largely based on identification of germline inactivating mutations in BRCA1/2 with an autosomal dominant inheritance Molecular Pathology in Clinical Practice, Table 24-2 Page 11 of 18 o It is rare, but breast cancer predisposition can be caused by mutations in genes associated with other syndromes, including Li-Fraumeni (TP53), Cowden (PTEN), and PeutzJeghers (STK11/LKB1) o Inherited mutations in BRCA1 and BRCA2 genes increase the lifetime risk for developing breast cancer (40-80%) and the lifetime risk of developing ovarian cancer (11-40%) vii. Peutz-Jeghers Syndrome (PJS) • Autosomal dominant inheritance pattern • PJS patients have characteristic polyps of the GI tract with low malignant potential, hyper-pigmented mucocutaneous lesions, and a family history o PJS patients are predisposed to a wide variety of neoplasms that arise in different organs • The average age of presentation is 29 years old • Mutations found throughout the STK11/LKB1 gene have been associated as causative genes for PJS Page 12 of 18 o Mutation types include truncating mutations, missense mutations, and deletions B. SOMATIC CANCERS 1. Role of the Environment Cancer is a combination of genetic and environmental factors. Most of human cancer is environmentally induced. Environmental carcinogens can be chemical or physical and certain viruses can also cause cancer. Examples of known environmental carcinogens (some chemical and some physical) include UV radiation, ionizing radiation, polycyclic aromatic hydrocarbons, aromatic amines from the dye industry, alkylating chemicals, and asbestos. The metabolic activation of carcinogens results in reactive electrophiles that bind to DNA and induce mutations. The DNA damage caused by carcinogens has been conclusively linked to cancer initiation and progression. Do not memorize the items in this Common Chemical Carcinogens table. This is for your reference to all the types of commonly occurring chemicals that can contribute to cancer. Page 13 of 18 2. Role of Oncogenic Viruses Viruses have been isolated that cause cancer in many species, from birds to rodents, to humans. Oncogenic viruses can be RNA or DNA viruses. Viruses can cause oncogenesis in a number of different ways. In some cases, the viruses may produce an oncogene that does not have a counterpart in the host-cell genome that promotes activation of host proto-oncogenes (this concept was demonstrated by Drs. Bishop and Varmus). The viruses may produce a protein that promotes proliferation of a particular population of cells within the host. Chronic inflammation may lead to increased cell proliferation within the infected organ and the result of increased proliferation is the accumulation of new mutations that may lead to tumorigenesis. An example is HPV, The HPV virus produces viral proteins that interact or bind to host proteins that then lead to a loss-of-function of the tumor suppressor genes p53 and pRb. The HPV protein E6 binds to the host cell’s E6AP ubiquitin ligase that leads to binding of a trimer with p53 resulting in p53 proteolytic degradation. The HPV protein E7 binds directly to pRb resulting in its degradation. There are three prophylactic vaccines on the market that are directed against some of the high-risk HPV types with oncogenic potential. Page 14 of 18 They protect against infection with these HPV types and are estimated to prevent up to 90% of HPV-linked cancers. Viruses and Human Cancer (you do not need to memorize this table) RNA Virus Cancer Human T-lymphotrophic virus type 1 (HTLV-1) Adult T-cell leukemia Mechanism The viral protein Tax is a transcriptional activator that activates the proto-oncogenes c-sis and c-fos, altering the cell’s normal control on cellular proliferation and leading to transformation. The viral protein Tat is a transcription factor that activates transcription of interleukin-6 (IL-6) and IL-10, which are normally involved in proliferation of T-cells. Tat can also be released from the infected cells and promote angiogenesis, acting as an angiogenic growth factor. As the liver tries to replace scar tissue with new cells, the higher rate of proliferation increases the changes of mutations occurring. Mechanism Due to chronic HBV inflammation promoting tumorigenesis. Non-Hodgkin lymphoma HIV Kaposi sarcoma Hepatitis C (HCV) DNA Virus Hepatitis B (HBV) Epstein-Barr Virus (EBV) DNA Virus Human papillomavirus (HPV) Herpesvirus (HHV-8) Liver Cancer Cancer Hepatocellular carcinoma B- & T-cell lymphomas, Hodgkin disease EBV encodes a Bcl-2 protein that restricts apoptosis of the infected cells promoting cell survival. Cancer Cervical, oropharyngeal, anal, vaginal, and vulvar cancer Kaposi sarcoma Mechanism HPV synthesizes 8 different proteins, but the oncoproteins E6 and E7 have been shown to inactivate the tumor suppressor proteins Tp53 and pRb, leading to a loss of cell cycle control and DNA damage control. E6 binds to the host cell’s E6AP ubiquitin ligase that leads to binding of a trimer with p53 resulting in p53 proteolytic degradation. E7 binds directly to pRb resulting in its degradation. Squamous cell = squamous cell carcinoma Gland cells = adenocarcinomas HHV-8 encodes for a number of mimics of human genes, including some with immunologic and angiogenic properties. One example is the encoded vcyclin protein, which is the viral homologue to host cyclin D (normally involved in deregulation of cell cycle progression). The v-cyclin-CDK6 protein combination mediates phosphorylation and subsequent downregulation of Rb and inhibition of p27, blocking G1 cell cycle arrest. C. PRECISION MEDICINE IN ONCOLOGY 1. Classic Cytotoxic Therapies Before the advent of targeted therapies, the nonsurgical cancer treatment included chemotherapy or radiation. Chemotherapies use drugs called cytostatics that aim to stop cancer cells from continuing to divide uncontrollably. However, these drugs target not only cancer cells, but any cells that are dividing rapidly and therefore are usually associated with a number of very uncomfortable Page 15 of 18 side effects. The most common rapidly dividing cells naturally in the adult human body include blood-producing cells, hair cells, and the cells of the mucous membranes of the mouth and throat area and of the digestive system. Adjuvant chemotherapy is performed after surgery and aims at cancer cells that may have been left behind in the body. Neoadjuvant chemotherapy is done before the surgery. Palliative chemotherapy is done when it is no longer possible to remove all of the tumor cells and the goal is reduce the growth of the tumor and lessen symptoms. There is some chemotherapy that works in areas where radiation has been applied. Radiation therapy uses high-energy particles or waves, such as xrays, gamma rays, electron beams, or protons, to destroy or damage cancer cells. Radiation works by creating small breaks in the DNA in the cells affected by the radiation and these breaks prevent the cancer cells from dividing and also causes them to die. As with chemotherapy, radiation is not selective for cancer cells, but the treatment can be precisely localized and therefore the damage can be controlled. 2. Molecular Profiling Molecular Profiling involves looking at a variety of cancer-related genes or genome-wide profile of deletions, insertions, translocations, and amplifications to assist with diagnosis, prognosis, or treatment regime design. Two common methods to address genome-wide molecular profiling are by looking at the gene expression profile (microarray) or sequencing (next-generation sequencing). Other common methods used for assess gene mutations include PCR and Sanger Sequencing. Additional common methods used to assess translocations and amplifications include FISH and karyotyping. 3. Targeted Therapies If you recall, oncogene activation is a phenomenon where the survival and growth of a cancer cell is dependent on an activated oncogene or the inactivation of a tumor suppressor gene. Activated oncogenes are often the target for targeted therapies by directly blocking the aberrant function of the cancer cells. In other words, many of these targeted therapies that focus on activated oncogenes will only kill the activated activity of these tumor cells. For example, the first tyrosine kinase inhibitor (TKI) on the market was imatinib (Gleevec). This drug is a highly effective inhibitor for a large number of tyrosine kinases, but was originally FDA approved for the BCRABL fusion protein, created by the t(9;22) translocation in chronic myeloid leukemia. Although many of these drugs are very effective in initially treating tumors, many of the tumors will eventually develop resistance to the targeted therapy over time. This is not so surprising because Page 16 of 18 tumor cells are highly mutable so they can acquire resistance mutations and these mutations may interfere with the way the targeted therapy interacts with the tumor cell or else it may assist the tumor cell in focusing on a different pathway for oncogene addiction. Table 15-5: Cancer Treatments (adapted from Thompson and Thompson) Tumor Type Breast Cancer NCSLC GIST Drive Gene and Mutation amplified HER2 activated EGFR Translocated ALK or ROS1 activated KIT or PDGFRA FDA-approved Targeted Therapy trastuzumab gefitinib Mechanism of Action Anti-HER2 antibody TKI Crizotinib TKI imatinib, nilotinib, and dasatinib TKI activated MEK trametinib activated BRAF vemurafenib Melanoma Serine-threonine kinase inhibitor Serine-threonine kinase inhibitor 4. Immunotherapies Cancer cells are the body’s own cells and therefore they are sometimes not recognized by the immune system because they have developed methods to avoid detection or shut down an immune response. Checkpoint inhibitor immunotherapies work by disrupting the cancer cell’s signals and expose them to the immune system for attack. Cytokines are protein molecules that can help regulate and direct the immune system. In cancer treatment cytokines can be produced in the laboratory and injected in large doses in a patient to induce an immune response. There are some early studies looking at how the combination of these two immunotherapies might boost or re-boot a patient response. Page 17 of 18 It is interesting to note that there are some molecular markers in the patient’s tumor that can provide insight into how the patient may or may not response to immunotherapy. Many immunotherapies focus on tumors with defects in the DNA repair machinery and so microsatellite instability (MSI) testing can be used to look for MSI-high patients who are more likely to respond. Also, tumors with mutations in the MMR genes, discussed in Lynch syndrome, are also more likely to respond to immunotherapy. In non-small cell lung cancer (NSCLC), the presence of an STK11 mutation with a KRAS mutation is indicative that a patient will be less likely to respond to immunotherapy. The use of molecular profiling in patients undergoing immunotherapy treatments is likely to lead to more insights into how the molecular profile affects response to immunotherapy. D. LABORATORY TESTING FOR ONCOLOGY 1. Familial Testing When looking for a germline mutation, the laboratory can test any cell in the body since all the cells should have the mutation. Therefore, most of the time when testing for hereditary mutations a blood sample will do well to look for a mutation. Also, since the mutation will occur with allele frequency of either 50% (heterozygous) or 100% (homozygous), the testing does not require that many copies of the DNA be tested, also referred to as depth. 2. Somatic Testing In the case of somatic testing, we are usually referring to rare mutations. We can exclude polymorphisms because if the population carried the mutation at greater than 1% frequency, then the incidence of the cancer caused by the mutation would also be greater than 1%. Since the focus is on rare mutations and the mutations only occur in the cancer, it is important to focus our studies in the cancer while avoiding as many of the normal surrounding cells as possible. Also, since tumors are heterogeneous and clonal in nature, not all of the cancer cells are likely to have the exact same mutation profile, so the sensitivity and limit of detection of the test must be high and low, respectively, in order to focus on detection of these rare variants. Page 18 of 18