Genetics 10 - Cancer & Genomic Medicine Flashcards
Nature of most cases of cancer
Sporadic
< 10% of all tumours result from a familial disposition
Still a GENETIC disease
Cancer
General term for all malignant neoplasms
Malignant
when it grows independently of control mechanisms, being capable of transcending tissue boundaries, growing invasively, and metastasizing
Basal cell carcinoma
Very low metastatic potential and may grow and filtrate surrounding tissue without metastasizing for many years
Carcinomas
develop from epithelial tissue (e.g., skin, intestinal epithelium, bronchial epithelium, and the epithelium of the glandular ducts such as the mammary glands or pancreas)
Sarcomas
originate from mesenchymal tissue (e.g., connective tissue, bones, muscles)
Leukaemias and lymphomas
malignant diseases of the haematological and lymphatic systems
How do most cancers develop
Through progressive accumulation of various mutations within a cell
These genetic changes are typically acquired somatically, although some can be transmitted through the germ line and are present at birth in every body cell
Protooncogenes
Genes that, through (dominant) activating mutations, can be turned into oncogenes
Oncogenes facilitate malignant transformation by synthesis of structurally altered or defective proteins
Tumour suppressor genes
Genes that are relevant for the regulation of growth, repair, and cell survival, with malignant transformation supported through (recessive) loss-of-function mutations on both copies of the gene
They typically include DNA repair genes that are responsible for detecting and repairing genetic damage within a cell
Malignant transformation
The change from controlled to uncontrolled growth of a cell that is caused by mutations in oncogenes or tumour suppressor genes
Cancer is the result of
accumulation of several genetic and chromosomal changes
Tumour progression model - adenoma carcinoma sequence
Explains impact of a succession of different gene defects on tumour development
(normal tissue → adenoma → carcinoma in colon takes 10 years)
uncontrolled growth of a tumour
disruptions in intracellular, as well as intercellular, processing of information
Cell proliferation and cancer?
Not from cell proliferation
Question of balance between cell division and growth on 1 side
and apoptosis on the other
Differentiation of a malignant tumour
A malignant tumour tends to be less differentiated than its tissue of origin
How do oncogenes develop
from protooncogenes through hypermorphic mutations that result in gain of function
mutations are mostly missense - cause permanent activation or altered function of the gene product (qualitative changes)
Translocations and protooncogene
translocations can turn a protooncogene into an oncogene by generating a fusion gene with novel function and/or placing it under the control of a new, constitutively active promoter, which might trigger abnormal expression with regard to organ system or developmental stage
2nd way in which protooncogenes can be multiplied
Amplification
Increased gene copy numbers and thus more gene products in cell - quantitative
Intracellular dominance of oncogenes
Oncogenes are dominant at the cellular level, which means that activation or overexpression of one single allele is sufficient to result in a change of the cell’s phenotype
What are typical protooncogenes involved in
pathways that regulate cellular growth, cell proliferation, and the cell cycle
receptor tyrosine kinases
growth factors and their receptors
components of intracellular signaling cascades
proteins that regulate the cell cycle
RAS genes code for
guanosine triphosphate (GTP) binding proteins that have a crucial regulating function for several important signaling cascades in the cell
K-RAS mutations
90% of all pancreatic carcinomas
50% of all colon cancers
N-RAS
30% of all AML - Acute Myeloid Leukaemia
HER2/neu
Receptor tyrosine kinase
Important protooncogene in breast cancer
Philadelphia chromosome, t(9;22)q(34;11)
example of a chromosomal translocation resulting in activation of a protooncogene is the translocation between chromosomes 9 and 22
translocation causes a fusion of the BCR and ABL genes, leading to a fusion protein, BCR-ABL, and constitutional activation of the ABL tyrosine kinase
promoters for genes of the immunoglobulin chains
chromosome 14, 22 and 2
Result of translocation of protooncogenes into chromosomal regions that are under the control of promoters for genes of the immunoglobulin chains
uncontrolled, constitutive expression
MYC protooncogene
Burkitt lymphoma
Chromosomal translocations as causes of malignant diseases
Prognostic importance of tumour-specific balanced chromosome translocations
They are found only in tumor cells and thus represent somatic mutations
A cytogenetic analysis of the abnormal cell line should be included in the standard workup of most leukemias and lymphomas and may supply information on treatment strategies
Most common cytogenic change in paediatric Acute Lymphoblastic Leukemia
t(12;21)(p13;q22) translocation, which generates a TEL-AML1 fusion gene
BCR-ABL fusion gene and age
it is present in 5% of children, 35% of adults, and more than 50% of individuals over 60 years of age with ALL
particularly malignant form of the disease
median survival time of < 9 months when treated with conventional ALL regimens because of high early relapse rate
Most familial cancer predisposition syndromes result from
mutations in tumour suppressor genes in which loss of function favors development of a tumour
Products of TSGs
inhibit cellular growth, proliferation, or cell cycle progression (gatekeeper genes)
ensure genetic stability, for example, through DNA repair (caretaker genes)
How are oncogenes activated
Mutations on a single allele
How is a tumour-promoting phenotype associated with tumour suppressor genes is triggered
Inactivating mutations in BOTH ALLELES
these mutations are therefore recessive on a cellular level
Frequent TSG mutations
null mutations that cause complete absence of a functional product, such as small frameshift deletions or nonsense mutations that cause aborted protein synthesis
Unclassified variants
Missense mutations
Variant of unknown significance
A genetic variant identified in a patient with a particular disease or a suspected disease predisposition that may or may not be of functional importance
2-hit hypothesis of cancer development
Cancer development involves two successive mutations that affect the two alleles of a tumour suppressor gene
In familial cancer disposition syndromes, a mutation on one allele is inherited, and only one additional hit is required for cancer development
Sporadic retinoblastoma
did not inherit a mutation and require two independent somatic mutations affecting the same cell
2 hits required
congenital - one of the mutations is already present - a somatic mutation is likely to occur in at least one relevant cell, the disease occurs almost inevitably and much earlier in constitutional mutation carriers than in noncarriers - Frequently, secondary tumours can develop independently (e.g., osteosarcoma and leukemia)
What is seen in constitutional tumour predisposition syndromes, one mutated allele derives from the parental germ line so that offspring will have only one wild-type allele in all of their body cells (first hit)
1 mutated allele derives from the parental germ line so that offspring will have only one wild-type allele in all of their body cells (first hit)
Each additional inactivating mutation of the second (wild-type) allele causes loss of function of the respective gene product within the affected cell (second hit)
Recessive = dominant
Although inactivation of tumour suppressor genes reflects a recessive mechanism at the cellular level, the associated cancer predispositions are inherited as (autosomal) dominant disorders
Incomplete penetrance of an autosomal dominant tumour predisposition syndrome
Patients who never experienced a 2nd hit in the relevant organ in their lifetime
hence would not develop any tumour
Where is this paradox of a a dominant disorder with a recessive pathomechanism at the cellular level apply to
All conditions where abnormal functioning of a single cell is sufficient for the development of clinical symptoms
Necessity for modification of 2-hit hypothesis
2nd hit in 2nd allele did not necessarily have to involve a DNA change
Epigenetic processes, such as DNA hypermethylation, can also account for the inactivation of an allele of a tumour suppressor gene
Since the methylation status of a gene remains the same throughout all mitotic cell divisions, the effect resembles that of a true alteration of the DNA sequence
DNA repair genes
Inactivation of DNA repair genes does not immediately trigger abnormal cellular growth or differentiation
However, it causes failure to identify and repair mutations in the entire genome => increase in mutation rate
Has an impact on protooncogenes as well as other tumour suppressor genes/gatekeeper genes
accumulation of mutations is a decisive factor in tumour progression, inactivation of DNA repair genes significantly accelerates malignant transformation
Lynch Syndrome
Hereditary tumour predisposition caused by mutations of DNA repair genes
Due to mutations in DNA mismatch repair genes e.g. MSH2 or MLH1
Cause of functional loss of 2nd allele of TSGs
How can it be recognised
not caused by a point mutation (or epigenetic changes) but by larger deletions or chromosomal alterations that result in the total loss of that gene copy
Through molecular genetic analysis of Loss of Heterozygosity - LOH
Familial retinoblastoma mutation
Disease causing point mutation in RB1 gene - all normal cells show heterozygosity for that mutation, tumour tissue from the same individual will only reveal the mutated allele
What happens in the case of deletion
Describe the mutation
Normal allele is absent and hence there is a loss of heterozygosity
mutation is really hemizygous as the wild type allele is not amplified by polymerase chain reaction (PCR) due to a large deletion or possibly loss of entire chromosome
most frequent mechanism for a second hit in heritable tumour predisposition syndromes
LOH
Often seen as part of the malignant progression of sporadic tumours
Sporadic retinoblastomas
most commonly unifocal and unilateral
The average age at diagnosis is 24 months
60% of RBs
Hereditary retinoblastomas
more often multifocal and bilateral
The average age at diagnosis is 15 months
40%
Clinical mainfestations of retinoblastoma
Leukocoria - abnormal white reflextion in the iris of the affected eye
Strabismus - accompany/antedate the leukocoria
Visual detoriation - not noticed in small children
Less common sympotms = uveitis, glaucoma, hyphema (blood in anterior chamber of eye), proptosis, pain
Genetic basis for retinoblastoma
RB1 gene on chromosome 13q14.1-q14.2 is crucial in the pathogenesis of retinoblastoma
Classic tumour suppressor gene
RB1 gene on chromosome 13q14.1-q14.2
In order for tumour to develop (RB)
inactivation of both RB1 alleles in a retinoblast cell is necessary in order for a tumor to develop
Sporadic vs hereditary RB
In the case of hereditary retinoblastoma, one RB1 germline mutation is already present in all cells of the body
A retinoblastoma develops when a “second hit” inactivates the second RB1 allele in a retinoblast cell (loss of heterozygosity)
contrasts with the sporadic form of retinoblastoma, in which two independent somatic events must occur in the same cell (or successively in the same cell line) in order for a tumor to form
Family history of RB
only 10% of children with RB have a +ve family history
Other cases of hereditary retinoblastoma are based on de novo mutations in the parental (usually paternal) germ line
Patients with a hereditary RB1 mutation have a greater than 95% chance of developing retinoblastoma, despite the fact that they inherit only one mutated allele of the tumor suppressor gene, which is recessive at the cellular level
Mode of inheritance of hereditary RBs
autosomal dominant mode of inheritance
Mutations of RBs
Mutations that lead to a premature stop codon (nonsense, frameshift or splice mutations)
Therapy and management of RBs
Generally treated by enucleation of affected eye
+ adjuvant radiotherapy if necessary
Treatment of tumours of moderate size
radioactive plaque therapy (I-125 brachytherapy, which involves suturing of a radioactive plaque to the eye wall at the apex of the tumor)
Treatment of very small tumours
Laser ablation
Cryotherapy
Treatment for bilateral RBs
the more severely affected eye is enucleated and the contralateral eye is treated with radiation
Risk of RBs
increased risk of other primary ocular tumors and the potential for contralateral disease
Patients with the hereditary form of retinoblastoma are at increased risk for developing other malignant tumors including osteosarcomas
soft tissue sarcomas
melanomas
pinealomas
Li-Fraumeni Syndrome mode of inheritance
Autosomal dominant inheritance
How is Li-Fraumeni Syndrome characterised
increased risk of developing many types of cancer: soft tissue sarcomas, breast cancer, leukemias, osteosarcomas, melanomas, and tumors of the colon, pancreas, adrenal cortex, and brain
1/2 of affected individuals have a tumour by 30 yrs of age
90% at 65
high risk for developing multiple primary tumours
Genetic basis for Li-Fraumeni syndrome
50% - germline mutations of the tumor suppressor gene TP53 on chromosome 17p13.1
The tumor suppressor p53 serves a key function as gatekeeper of the genome
If DNA damage is recognized at the G1/S checkpoint of the cell cycle (i.e., before DNA replication), p53 induces cell death by activating the transcription of apoptosis-related genes
Small proportion - germline mutations in the CHEK2 gene on chromosome 22q
CHECK2 codes for a serine/threonine kinase that is part of the p53 signaling cascade
Familial Adenomatous Polyposis accounts for
how is it defined
0.5-1% of all colorectal cancers
Defined by presence of innumerable colorectal adenomas - detectable at least by late puberty in affected individuals
How do lesions associated with FAP develop
These lesions follow the adenoma–carcinoma sequence, starting as benign epithelial tumors and developing over a period of years into epithelial dysplasia and finally to colorectal carcinoma
What mutations are found in at least half of all malignant tumours
Somatic mutations of the TP53 tumour suppressor gene
What is the most commonly mutated gene in human malignancies
TP53
Classification of FAP
Obligate precancerous lesion
Apart from colon lesions, what do FAP patients also have
extracolonic manifestations
Attenuated FAP
variant of FAP in which the number of polyps is far less than 100
produces clinical manifestations much later than classic FAP, with colorectal cancer usually developing in the fifth decade
Attenuated FAP is still associated with a high cancer risk
Genetics and etiology of FAP
FAP and attenuated FAP are caused by heterozygous mutations in the APC tumour suppressor gene (adenomatous polyposis coli) on chromosome 5q21-q22
Lead to truncation of gene product
Mode of inheritance of FAP
Autosomal dominant mode of inheritance
1/4 of patients represent de novo cases
detailed family history should be taken in all patients with colon cancer
Analysis performed in individuals with a clinical diagnosis of FAP and in at-risk family members
molecular genetic analysis of the APC gene
What should be done if a single colorectal carcinoma is detected
the entire colon should be scrutinized for possible additional adenomas (complete colonoscopy) because at least one more adenoma is present in up to 30% of cases
Lynch Syndrome (Hereditary Nonpolyposis Colon Cancer - HNPCC) & colorectal cancers
5% of all colorectal cancers
Cardinal feature of Lynch Syndrome (HNPCC)
Familial occurence of colon cancer or certain other cancers at a relatively young age
Median age at onset of CR cancer in Lynch syndrome
45 yrs - cancers rarely occur before 25
Unlike sporadic cancers, where are Lynch Sydrome associated cancers located
frequently (in more than 50% of cases) located in the right side of the colon, and so cancers at that location are always suspicious for HNPCC
Risk of developing CR cancer
as high as 80% in male carriers of the HNPCC mutation
Female carriers have a 50% lifetime risk for colon cancer and a 60% risk of developing endometrial cancer
Extracolonic neoplasms that have been linked to Lynch Syndrome
include cancers of the stomach (10% to 20% lifetime risk), ovaries (12%), biliary tract (7%), small intestine (4%), urothelium, pancreas, and CNS
Mode of inheritance - Lynch Sydrome
heterogenous and generally has an autosomal dominant mode of inheritance
Mutations - Lynch syndrome
DNA mismatch repair system - Usually these involve mutations in the MLH1 or MSH2 genes
Other Lynch syndrome–associated genes are MSH6, PMS1, and PMS2 - Like other DNA repair genes, these are tumor suppressor genes
Inherited heterozygous mutations are not sufficient by themselves to produce a clinical phenotype
A “second hit” on the healthy allele is necessary to cause a complete loss of functional protein, resulting in defective DNA mismatch repair
Between what tumour entities is there a link
genetic risk factors - relatives of women with ovarian cancer also have a 30% to 60% greater risk of developing breast cancer
Genes associated with breast cancer
BRCA1
BRCA2
Risk factors for breast cancer
(While there are heritable disorders that predispose to breast cancer, the vast majority of cases have a multi-factorial etiology)
Menarche before 12 years of age
Menopause after 55 years of age
Having a first child after 30 years of age
Nulliparity
Postmenopausal obesity
Alcohol abuse
Hormone replacement therapy
Radiation exposure
proportion of breast cancers based on genetic predisposition
5-10%
BRCA1 and BRCA2
BRCA1 is located on chromosome 17q21 and BRCA2 on chromosome 13q21
Both genes are tumor suppressor genes that code for proteins involved in DNA damage repair and are involved in cell cycle control and regulation of other proteins of DNA damage response
Mode of inheritance of familial BC
autosomal dominant manner
What might molecular genetic testing of the tumour tissue in breast cancer demonstrate
a loss of heterozygosity (LOH) in the corresponding chromosomal region
Mutations associated with breast cancer
Nonsense and frameshift mutations are clearly associated with disease
Unclassified variants - cannot be used for susceptibility testing in other family members
Lifetime disease risk associated with Pathogenic Mutations in BRCA1 and BRCA2
Penetrance of BRCA1 and BRCA2 mutations
An individual who harbors a BRCA1 or BRCA2 mutation has an increased susceptibility to disease; however, the penetrance is not complete
Some carriers develop multiple primary tumors before 50 years of age, whereas other people with the same mutation do not develop a single cancer past age 70
The lifetime risk for breast cancer in mutation carriers is 85%, and more than half of affected women will develop breast cancer before age 50
Carriers of BRCA1 mutations have a significantly higher risk of developing ovarian cancer (44%) than carriers of BRCA2 mutations (27%)
BRCA1 and men
BRCA1 mutations are associated with an approximately threefold increase in the risk for prostate cancer but very rarely lead to male breast cancer
BRCA2 and men
a definite association exists between BRCA2 mutations and male breast cancer, with carriers facing a cumulative risk of 7% by the age of 70 years
BRCA2 overall
BRCA2 mutations show less association with prostate cancer but have been linked (in both sexes) to tumors of the pancreas, larynx, esophagus, colon, stomach, and biliary tract, and to melanomas
Diagnosis of breast cancer
If a tumour is found it is sized and graded and a biopsy is done if necessary and ER, PR and HER2 status assessed. Based on these results further tests may be ordered including the Oncotype DX molecular profiling test (if an early stage ER or PR positive and HER2 negative cancer)
when is genetic testing offered
to an individual affected with cancer, whose combined personal and family history of cancer, has a combined BRCA1 and BRCA2 mutation carrier probability of 10% or more
This equates to a Manchester Score of ≥ 15
NCCP criteria for diagnostic BRCA1/2 testing
Targeted mutation analysis is offered to
women of Ashkenazi Jewish heritage (of Jewish or Polish ancestry)
It includes mutations known to be at greater frequencies because of founder effects
Comprehensive analysis includes full sequence analysis of BRCA1 and BRCA2 and testing for specific large genomic rearrangements of BRCA1
What should be offered to women with documented BRCA1 and BRCA2 mutation
should be offered a prophylactic mastectomy and oophorectomy as a preventive measure
`2 types of mastectomy
Subcutaneous mastectomy (approximately 5% residual tissue)
Complete mastectomy (approximately 1% residual tissue) - women over 35 with no desire for future pregnancy
3% residual risk of peritoneal cancer
Precision/personalised medicine
use of each person’s unique combination of genetic (as assessed by genomics) and environmental risk factors to make predictions about his or her disease risk and response to various treatments
MOA of NGS (Whole Genome Sequencing, Whole Exome Sequencing, RNA sequencing)
rely on massively parallel sequencing (sequencing many overlapping parts of the genome at the same time)
powerful tools for discovering what genes and sequences are associated with disease
WES
makes it possible to cost-effectively determine nearly all the coding variation in an individual human genome, which sums to less than 2% of the genome, in a single experiment
Exons are targeted and then sequenced using this technique, which has become a powerful new approach for identifying genes underlying mendelian conditions, particularly in circumstances where conventional approaches have failed
WGS
analyses the entire genome, coding (exons) and non-coding (introns and intergenic regions) DNA
As the cost of wholegenome sequencing continues to drop, it will likely become the preferred strategy for gene discovery
RNA-seq
used for expression profiling of normal versus disease tissues, and is often used in personalised cancer medicine
RNA-sequencing has been used to develop microarrays for tumour expression profiling
Bioinformatics
refers to an interdisciplinary field that develops methods and software tools for understanding biological data
It involves using computers, statistics, maths and engineering to analyse and interpret biological data
Exome sequencing
- Genomic DNA is randomly sheared to create a library of DNA fragments that are flanked by adapter sequences (not shown)
- The library is enriched for sequences corresponding to exons (dark blue fragments) by hybridization capture
- In the capture procedure, also known as “pull down,” the fragments are hybridized to biotinylated DNA or RNA baits (orange fragments) in the presence of blocking oligonucleotides that are complementary to the adaptors (not shown)
- The hybridized DNA fragments are amplified by PCR and rapidly sequenced using high-throughput techniques
- The end result is the DNA sequence of the exons in the genome
Cancer genomics
study of the DNA-associated changes that accompany cancer with the overall goal of better preventing, detecting, diagnosing, and treating common cancers
How have classification schemes of cancers been developed
(leukemia, lymphoma, and cancers of the breast, lung, colon, and brain)
use of genome-wide gene expression analyses (using RNA-seq or RNA microarray) to provide a snapshot of gene activity within a tumor at a given point in time
TNM system
often difficult to predict the prognosis of cancer patients based on traditional phenotypic information such as the type of tumor (T), whether the cancer is found in nearby lymph nodes (N), and evidence of metastasis (M)
Not predictive of prognosis or treatment response
cancers that are easily confused
Burkitt lymphoma vs. diffuse large B-cell lymphoma
Gene-expression profiling can help to distinguish between cancers that are easily confused
What measurements are mandatory for all patients with invasive BC
measurement of ER, PR and HER2 is mandatory
MammaPrint assay and Oncotype DX
generates a 70 gene predictive signature for distant recurrences after surgery in early-stage breast cancer patients
It can potentially save many patients the trauma of chemotherapy
In Ireland a similar assay Oncotype DX is used and does expression profiling of 21 genes by RT-PCR.
Impact of gene-expression profiling of cancers
helping to improve the classification of different types of tumors and may help to guide therapy (personalised medicine)
