Week 7

Question 1

gene

Answer 1

a

Question 2

locus

Answer 2

a segment of DNA at a specific location is called a locus; if the segment contains a gene then the DNA segment is the locus for the gene

Question 3

allele

Answer 3

alternative variants of a gene are called alleles

Question 4

polymorphism

Answer 4

variant alleles are said to show polymorphism and thus variant alleles are also referred to as polymorphic alleles or polymorphisms; some of these affect disease susceptibility (vs wildtype)

Question 5

wild-type

Answer 5

for most genes there is a single prevailing allele, present in the majority of individuals called the wild-type allele; the other versions are called variants or mutants

Question 6

mutation

Answer 6

for most genes there is a single prevailing allele, present in the majority of individuals called the wild-type allele; the other versions are called variants or mutants

Question 7

genotype

Answer 7

genotype refers to either an entire set of alleles in a genome or the set of alleles at a specific locus

Question 8

phenotype

Answer 8

phenotype refers to the observable expression of a genotype as a morphological, clinical, cellular or biochemical trait

Question 9

heterozygous

Answer 9

heterozygous means the two alleles are functionally different;
a special situation is when an individual has only one allele of a gene, this is called hemizygous;
compound heterozygotes are individuals with two heterogeneous recessive alleles at a particular locus that can cause genetic disease in a heterozygous state

Question 10

homozygous

Answer 10

homozygous means an individual’s two alleles are functionally identical at a specific locus

Question 11

kindred

Answer 11

a kindred is the extended family depicted in the pedigree

Question 12

proband

Answer 12

the proband is the first affected person who is brought to clinical attention (and there can be multiple probands); all other family members are analyzed in relation to the proband; there is another term, consultand, that refers to the person who brings the phenotype to clinical attention (this can be an affected or unaffected individual)

Question 13

consanguineous

Answer 13

couples who share one or more ancestors in common are consanguineous

Question 14

fitness

Answer 14

fitness is a genetics term that refers to the measure of the impact of a condition or genotype on reproduction and is defined by the number of offspring of affected individuals who survive to reproductive age, compared with an appropriate control group

Question 15

Given a patient’s family history, be able to construct a pedigree.

Answer 15

a pedigree is a graphical representation of the family tree, using standard symbols

females: circles
males: squares

Question 16

Given a pedigree, predict a disease’s mode of inheritance

Answer 16

be able to do this

Question 17

autosomal

Answer 17

autosomal disorders generally affect males and females equally

Question 18

recessive

Answer 18

defined as a phenotype only expressed in autosomal homozygous mutants or X-linked males (who are X hemizygotes) but not in heterozygotes
most recessive diseases involve a loss-of-function, such that mutations in both alleles eliminates gene function, e.g., of a gene encoding for an enzyme
heterozygote carrier parents are usually phenotypically normal because they make enough gene product from one wildtype allele to prevent disease

Question 19

dominant

Answer 19

a phenotype expressed in both homozygotes and heterozygotes is considered dominant
when both homozygotes and heterozygotes show an identical severity of phenotype it is called pure dominant, but this rarely happens
more commonly a disease is more severe in homozygotes, a situation called semidominance or incomplete dominance
on occasion when two different variant alleles are expressed together they are considered codominant (e.g. ABO blood group)

Question 20

X-linked

Answer 20

In X-linked disorders, males are hemizygous for genes on the X chromosome (having a single X chromosome), thus it is far more common for males to develop X-linked recessive diseases, while females can be heterozygous or homozygous for X chromosome genes
females randomly inactivate one of their X chromosomes in each cell, thus even if they inherit a dominant X-linked mutant gene, the phenotype may only be expressed in a subset of cells, resulting in mosaicism; mosaicism also is seen in X-linked recessive diseases where females demonstrate an attenuated phenotype compared with males

Question 21

Consider factors such as penetrance, allelic heterogeneity, sex limited phenotypes, and effects on fitness in interpeting pedigrees.

Answer 21

a

Question 22

penetrance

Answer 22

in some diseases and families an individual who inherits the disease causing genotype may not show the same or customary phenotype as others in the family; this is usually due to reduced penetrance or variable expressivity
penetrance is the probability that a mutant gene will have any phenotypic expression; when the percentage of individuals demonstrating some disease phenotype is less than 100% the mutant gene is said to demonstrate reduced penetrance
expressivity is the severity of expression of the phenotype among individuals with the same disease causing genotype; when the severity of the disease differs in people who have the same genotype the phenotype Is said to have variable expressivity

Question 23

allelic heterogeneity

Answer 23

many loci contain multiple mutant alleles in a population
for example, more than 1400 mutations have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene among patients with cystic fibrosis; in some cases the different mutations cause the same clinical disease phenotype but in other cases mutations can cause either a more severe phenotype (pancreatic insufficiency, severe progressive lung disease, congenital absence of the vas deferens in males) or an attenuated phenotype (only the lung disease or only an abnormality in the male reproductive tract), thus the CFTR mutations can be ordered along a continuum of severity; this phenomenon is called allelic heterogeneity
PKU (phenylketonuria) is another example of a disease that demonstrates allelic heterogeneity
for many autosomal recessive diseases, affected individuals carry two different mutant alleles (compound heterozygotes), and the particular combination of mutant alleles can have a large impact on disease severity

Question 24

locus heterogeneity

Answer 24

many disease phenotypes can be caused by mutations in distinctly different genes, thus making it difficult to determine the causative gene, with important implications for therapy; this phenomenon is called locus heterogeneity
an example is retinitis pigmentosa, a common cause of photoreceptor degeneration, has been show to have autosomal dominant, autosomal recessive and X-linked forms, all associated with different mutant genes; overall, more than 70 genetic diseases manifest themselves as retinitis pigmentosa
hyperphenylalanemias (which include PKU) is another example of a phenotype that can be caused by mutations in different genes

Question 25

phenotypic heterogeneity

Answer 25

in some genes different mutations in the same gene cause completely different diseases; this phenomenon is called phenotypic (or clinical) heterogeneity
an example is the RET gene which encodes a receptor tyrosine kinase
one mutation in Ret causes a dominantly inherited failure of development of colonic ganglia, leading to defective colonic motility and chronic constipation (Hirschsprung disease)
another Ret mutation causes a dominantly inherited cancer of the thyroid and adrenal glands (multiple endocrine neoplasia type 2A and 2B)
a third Ret mutation can cause both Hirschsprung disease and the endocrine cancers

Question 26

Discuss how allelic variation in a population can lead to different disease susceptibilities; e.g. alpha1-antitrypsin deficiency.

Answer 26

alpha1-antitrypsin deficiency:
 major serum protein that inhibits proteolytic enzymes; major target is leukocyte elastase, which can damage lung connective tissue if not down-regulated
 5 major alleles (M1,M2,M3,S, and Z) that differ in the amount of effective protein
 people with ZZ genotype make only 15% of normal amount of protein and are susceptible to early onset emphysema and other diseases
 allele frequencies vary by ethnicity with Z frequency highest in Caucasians (especially Danes)

ZZ causes increased risk of death for smokers

Question 27

Describe evolutionary pressure to maintain disease causing alleles in a population; e.g. sickle cell anemia.

Answer 27

 S is an example of a deleterious allele that is maintained in a population because when heterozygous it increases reproductive fitness = concept of heterozygous advantage

Question 28

Given a population’s genotype frequency, calculate allelic frequency.

Answer 28

 just count the alleles: e.g., if a population has 20 AA, 10 Aa, and 5 aa - thus there are a total of 70 alleles, 50 of them are “A”, so the “A” allele frequency is 50/70 = 0.714; the “a” allele frequency must then be 1-0.714=0.286

Question 29

Given an allele frequency, use the Hardy-Weinberg equation to calculate a population’s genotype frequency.

Answer 29

p2 + 2pq + q2
for a two allele locus (A & a),with p2 as the probability of the AA genotype, q2 as the probability of the aa genotype, and 2pq as the probability of heterozygotes (Aa)

Question 30

Discuss the changes in protein function associated with “single gene mutation” diseases (loss of activity, increased activity, novel activity, change in spatial or temporal activity)

Answer 30

loss of function mutations
 largest category
 can occur in coding and non-coding gene regions
 can arise from a variety of mutations such as point (missense, nonsense,frameshift), deletions, insertions
 a few examples: -globin (thalassemias), PAH (PKU), p53 (cancer)

gain of function mutations
 can arise from increased gene dosage or increased protein function
 Down’s syndrome is probably due to increased gene dosage
 achrondroplasia (short stature) is an example of increased function - single amino acid change results in overactivation of fibroblast growth factor receptor (FGFR)

novel properties
 example of sickle cell disease where sickle hemoglobin chains aggregate when deoxygenated, leading to deformation of red blood cells

inappropriate expression
 many examples in cancer: genes normally quiescent in adult animals are turned on, for example, developmental growth factors; cancers may also express a gene in a tissue where it is not normally expressed

Question 31

discuss environmental and genetic factors which can modify the penetrance and course of pathology for diseases caused primarily by defects in a single gene

Answer 31

 allelic heterogeneity: different alleles of the same gene cause varying disease severity; example of PAH
 locus heterogeneity: mutations in different genes can yield a similar clinical phenotype; for example, alteration in 5 different genes can cause hyperphenylaninemia
 modifier genes: people (even within families) with the same mutation can present dramatically different phenotypes due to the presence of modifier genes; classic example is ApoE4, if you carry 1 or 2 (worse) alleles of ApoE4 you are more susceptible to a range of neurological and neurodegenerative disorders

Question 32

Identify the inheritance pattern, genetic defects and molecular causes underlying I-cell disease

Answer 32

I-cell disease, an autosomal recessive lysosomal storage disease caused by a defect in protein trafficking; acid hydrolases which are required for lysosomes are not properly modified with glycoproteins (called mannose-6-phosphates)and get sent out of the cell instead of to the lysosomes

Question 33

Identify the inheritance pattern, genetic defects and molecular causes underlying Tay Sachs disease

Answer 33

Tray-Sachs disease, an autosomal recessive disorder caused by a buildup of GM2 ganglioside sphingolipids. Genetic defect is a mutation in hexA gene; hexA is ubiquitously expressed but only has a mutant phenotype in the brain; Tay-Sachs 100 times more prevalent among Ashkenazi Jews; 1 in 27 AJ carry a mutant hexA allele; homozygous infants are normal until 3-6 months, then gradual neurological problems begin, leading to death between 2-4 years

Question 34

Identify the inheritance pattern, genetic defects and molecular causes underlying cystic fibrosis

Answer 34

is cystic fibrosis (CF), an autosomal recessive disease which has an incidence of 1/2500 in Caucasians (1/25 carriers
 CF caused by mutation in the CFTR gene which encodes a chlorine channel located in the apical membrane of epithelial cells
 lungs & exocrine pancreas are two major organs affected
 most common mutation is 508F, a 3 bp deletion which eliminates a phenylalanine that causes misfolding of the protein
 508 mutation is more severe than others; another example of allelic heterogeneity

Question 35

Identify the inheritance pattern, genetic defects and molecular causes underlying phenylketonuria

Answer 35

 almost all enzymes are proteins; enzymes contain critical regions that can be disrupted by mutations
 example: PAH gene and the disease PKU. PKU patients accumulate phenylalanine in body fluids which can damage CNS
 PKU is a relatively common defect in newborns (1/2900) that is tested for at birth and treated with dietary modification(although adult mild retardation is common)
 PKU is an example of defect that occurs in one tissue (liver & kidney) but where the phenotype is manifest elsewhere (brain)

Question 36

Identify the inheritance pattern, genetic defects and molecular causes underlying hypercholesterolemia

Answer 36

 example: low-density lipoprotein receptor (LDLR), which is responsible for binding & internalization of LDL & cholesterol
 hypercholesterolemia is an autosomal dominant disorder arising from LDLR defects, and is common familial disease (1/500 as heterozygoytes) that is more severe in homozygotes
 LDLR deficiency in liver causes cardiovascular disease (atheromas)

Question 37

Identify the inheritance pattern, genetic defects and molecular causes underlying alpha1-antitrypsin deficiency

Answer 37

 major serum protein that inhibits proteolytic enzymes; major target is leukocyte elastase, which can damage lung connective tissue if not down-regulated
 5 major alleles (M1,M2,M3,S, and Z) that differ in the amount of effective protein
 people with ZZ genotype make only 15% of normal amount of protein and are susceptible to early onset emphysema and other diseases
 allele frequencies vary by ethnicity with Z frequency highest in Caucasians (especially Danes)

ZZ expression leads to drastically decreased survival rates for smokers

Question 38

Identify the inheritance pattern, genetic defects and molecular causes underlying Duchennes Muscular Dystrophy

Answer 38

Duchennes Muscular Dystrophy (DMD), an X-linked recessive disease caused by a mutation in the dystrophin gene; DMD occurs in 1/3300 live births; DMD causes progressive muscle deterioration from childhood on, leading to death by late teens; it is currently untreatable but gene therapy trials are underway
 dystrophin encodes for a huge protein (427 kD) with two functions: maintains muscle-membrane integrity, linking actin skeleton to the ECM; and it maintains synaptic junctions in the brain
 1/3 of DMD arise from new mutations: this rate is enhanced because of the size of the gene & because of higher mutation rates in sperm
 carrier mothers have no clinical manifestations but often show elevated creatine kinase levels

Question 39

Discuss strategies used to map the genetic causes and inheritance patterns of singe gene and complex genetic diseases.

Answer 39

a

Question 40

Association analysis

Answer 40

 unlike linkage analysis one starts with a candidate gene, suspects that a defect or polymorphism is responsible - and then looks in families and/or a population to determine if people with a disease are statistically more likely to carry a particular mutation or polymorphism
GWAS are a special form of association analysis on a mass scale
similar complications as linkage analysis
much more common in complex disease genetic analysis than monogenic diseases
resolution is much more precise than linkage analysis, as low as 10-50 kb (vs 10 MB)
 often today we use SNPs and SNP haplotypes to follow in families or a population

Question 41

linkage analysis

Answer 41

2 genetic loci are linked if they are transmitted together from parent to offspring more often than expected under independent inheritance
 linkage analysis is used to study families to determine if two genes demonstrate linkage when passed from one generation to another
 the closer two genes or markers are to each other the less likely they will be separated during meiotic recombination. The likelihood of separation is called the recombination frequency (RF). A RF of  50% means two genes or a gene and marker are unlinked; < 50% means they are linked
 RF of 1% = 1 centiMorgan (cM), unit of genetic distance that corresponds to ~ 2 MB of sequence

Question 42

sib-pair analysis

Answer 42

uses small families and asks whether affected siblings share specific gene alleles at a frequency high than expected by random chance
can also ask whether an affected offspring and unaffected sibling share or do not share parental alleles

Question 43

Discuss the implications of the clonal evolution hypothesis, stem cell hypothesis, and two hit hypothesis as they apply to the development, progression and clinical management of cancer.

Answer 43

be able to do this

Question 44

Provide an example of how gene expression profiling of cancers can identify patient subpopulations with differential survival and responses to therapy.

Answer 44

a

Question 45

Provide examples of how genetic polymorphisms in the human population in genes involved in drug metabolism have a profound impact on drug toxicity and efficacy.

Answer 45

a

Question 46

Describe five types of genetic polymorphisms and their use in genetic analysis.

Answer 46

(insertions, deletion, tandem repeat, single nucleotide polymorphisms, restriction fragment length polymorphisms)
we need markers that can distinguish between individuals and between carriers and non-carriers of a disease gene
 used to detect, map and clone disease genes
most common markers are DNA sequence variants

Question 47

Describe how single nucleotide polymorphisms, which may or may not affect protein structure, can be used as genetic markers.

Answer 47

It is known that pieces of DNA that lie near each other on a chromosome tend to be inherited together. This property enables the use of a marker, which can then be used to determine the precise inheritance pattern of the gene that has not yet been exactly localized.

Question 48

Describe how haplotype mapping and genotype wide association studies can identify genetic factors associated with disease susceptibility

Answer 48

if a disease mutation/polymorphism arises in an individual with a distinctive haplotype, it can be followed in a population without knowing the identity of the disease gene

Question 49

Compare and contrast the genetic events leading to Angelman’s syndrome and Prader-Willi syndrome, both caused by mutations at imprinted loci.

Answer 49

PW gene is maternally imprinted. When a deletion or other mutation occurs in the expressed allele no PW gene product is made and the result is PWS

AS gene is paternally imprinted. When a deletion or other mutation occurs in the expressed allele no AS gene product is made and the result is AS.

Question 50

Describe aspects of clinical cytogenetics

Answer 50

definition: study of chromosomes, their structure and their inheritance, as applied to medical genetics
aspects:
karyotyping
FISH

Question 51

Develop a plan for testing family members of a patient with a genetic disease (e.g. cystic fibrosis) and interpret results to make recommendations for the family.

Answer 51

 pedigree analysis to exclude or include a family member
 determine if the exact CFTR mutation is known (e.g., 508F), if so, take DNA from white blood cells and test using PCR or Dot-blotting
 if mutation is unknown, try to identify polymorphic disease-linked markers (VNTR or RFLP) and test individual
 if no markers available and mutation is unknown, test for 508F; if negative, advise patient of relative risk: 30% (non-508F)x risk based on pedigree

Question 52

restriction fragment length polymorphisms

Answer 52

allelic variant that abolishes or generates a restriction endonuclease recognition site or changes the size of an RFLP (insertion or deletion)
 use to distinguish between 2 chromosomes
 usually just a biomarker & not a cause of a dysfunctional gene
 can be analyzed by Southern blotting or PCR

Question 53

tandem repeat

Answer 53

VNTR (variable number of tandem repeats; also called simple sequence length polymorphisms (SSLPs)
 tandem repeats (e.g., CACA//CACA, grouped as microsatellites, make up a significant part of the genome, are usually not part of genes and thus are not conserved
 often polymorphic in size between chromosomes & individuals, thus can be used as biomarkers
 analyzed mainly by PCR

Question 54

single nucleotide polymorphisms

Answer 54

 most common polymorphism (1: 300-1000 bp); thus there are ~ 3 million SNPs between any two human genomes; overall ~ 11 million SNPs have been identified in humans
 true SNP must have at least 1% frequency (some studies set a 5% threshold); 7 million SNPs have a frequency of > 5% and 4 million SNPs have a frequency of 1-5%, with innumerable rare variants < 1%
 use of SNPs : polymorphic biomarkers and disease-association
 SNP chips can detect thousands of SNPs; prediction that there will be chips that can detect susceptibility to a wide range of diseases, especially complex genetic diseases such as cancer, type 2 diabetes, cardiovascular
 SNPs already useful in gene mapping & pharmacogenetics

Question 55

karyotyping

Answer 55

to detect a chromosomal abnormality one can start with large defects visible on whole chromosomes
karyotype = chromosomal complement of a cell, individual or species. It describes the microscopic morphology of chromosomes: relative length, centromere positions, other features
 to examine germline karyotypes requires creation of metaphase spreads from T-cells grown in cell culture

Question 56

FISH

Answer 56

 molecular cytogenetics
 multi-chromatic fluorescent probes can target chromosomes, chromosome regions or genes
 often used in genetic testing for diseases such as cancer, prenatal disorders
 combinations of FISH probes (spectral karyotyping or SKY)
 doesn’t require metaphase spreads, can be conducted on tissue sections

Question 57

cytogenetics in cancer

Answer 57

 cytogenetic changes common in advanced cancers
 aneuploidy is the most common
 translocations can disrupt tumor suppressor genes or activate oncogenes
 BCR-ABL = Philadelphia chromosome in chronic myelogenous leukemia (CML) is a classic example

Question 58

Down Syndrome

Answer 58

 usually caused by trisomy 21 which is lethal in 75% of fetuses
 most common chromosomal birth defect: 1/800 live births
 rate increases to 1/15 in women over 45 - testing is recommended for women > 35
 8-fold (1/100) risk of recurrence
 trisomy 21 usually caused by meiotic non-dysjunction in meiosis I (can also occur in II)
 disease likely caused by increased gene dosage
 Down’s gene has not been identified, but very recent evidence suggest that a miRNA may be responsible

Question 59

sex-linked disorders

Answer 59

 Trisomy X: 1/1000 females
 Klinefelter Syndrome: 1/1000 males, caused by extra copies of X chromosome
 XYY (males): 1/1000 males
 Turner Syndrome: 1/5000 females, caused by X deletion

Question 60

invasive techniques

Answer 60

 amniocentesis: removal of amniotic fluid transabdominally by syringe; fetal cells are cultured for diagnostic tests
 cordocentesis: removal of fetal blood from the umbilical cord, more often used when other methods have failed or are ambiguous
 chorionic villus sampling: biopsy of tissue from the villous area of the chorion, can occur 4-5 weeks before amniocentesis

Question 61

non-invasive techniques

Answer 61

 maternal serum screening (double, triple, quad)
 ultrasound
 radiography
 MRI

Question 62

maternal serum screening (MSS) techniques

Answer 62

first trimester: HCG (free -human chorionic gonadotropin) increase in Down Syndrome; PAPP-A (pregnancy associated plasma protein-A) decrease in Down Syndrome
second trimester: can detect alpha-fetoprotein (AFP), unconjugated estriol (uE3), inhibin and human chorionic gonadotropin (HCG)
 depressed levels of AFP (& uE3) and elevated levels of HCG and inhibin A are associated with Down’s syndrome
 increased levels of AFP are associated with neural tube defects such as open spina bifuda

Question 63

prenatal testing strategy for Down Syndrome

Answer 63

start with non-invasive 1st and 2nd trimester screening in combination; ultrasound plus HCG and PAPP-A in the 1st trimester, followed by AFP, uE3, inhibin, and HCG in the 2nd trimester; this results in ~95% detection rate for Down Syndrome
positive non-invasive test results justify confirmative invasive testing, e.g., amniocentesis and FISH on fetal cells
availability of genetic and reproductive counseling

Question 64

new born screening

criteria:

Answer 64

 treatment available if defect detected
 evidence that early detection is of treatment value
 physical exam cannot detect the defect
 test is reliable
 screening is cost effective

Question 65

Define neoplasia, and explain the differences between benign and malignant neoplasms.

Answer 65

Neoplasia means “new growth” and a new growth is called a neoplasm. Tumor refers to a swelling or mass, which may or may not be neoplastic (e.g. hematoma). However, the term tumor is most commonly used by clinicians to refer to a neoplasm (so clinicians typically use “nodule” or “mass” to describe lesions which could be tumors). Oncology is the study of tumors (neoplasms).

Best definition of neoplasm (from Robbins): “A neoplasm is an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues and persists in the same excessive manner after cessation of the stimuli which invoked the change.”

The underlying pathogenesis of neoplasia is that the stimulus causes genetic alterations in a single cell, which are then passed on to the progeny of the tumor cell and all subsequent tumor cells, allowing excessive and unregulated proliferation that becomes autonomous. Thus a neoplasm is a clonal proliferation, forming a tissue mass that results from excessive and unregulated cell proliferation due to the genetic alterations.

Neoplasms are characterized as either benign or malignant. The key difference between a benign vs malignant neoplasm is that a benign neoplasm cannot spread to other tissues (does not metastasize), whereas malignant neoplasms have the capability to metastasize. Benign neoplasms are also generally not locally invasive (there are exceptions), whereas malignant neoplasms tend to be locally invasive and destroy adjacent structures (there are exceptions). The general term cancer (Latin word for crab) refers to malignant neoplasms.

Benign neoplasms are generally well differentiated and have a low mitotic rate; malignant neoplasms generally are less well differentiated and have a higher mitotic rate, and may show atypical mitotic figures. However, amongst malignant tumors exceptions occur, and some malignant neoplasms can be well differentiated with a low mitotic rate.

see chart on slide 17

Question 66

hypertrophy

Answer 66

increase in the size of cells, resulting in increase in the size of an organ (e.g. uterus in pregnancy).

Question 67

hyperplasia

Answer 67

increase in the number of cells, which can form a tissue mass. However, the proliferative process, whether physiologic or pathologic, remains under control because there are no mutations in the genes that regulate cell division. Hyperplasia will regress once the stimulus is removed.

Question 68

metaplasia

Answer 68

reversible change in which one differentiated cell type is replaced by another.

Question 69

hamartoma

Answer 69

non-neoplastic disorganized aggregate of mature tissues indigenous to the site of origin (some are actually neoplasms!)

Question 70

dysplasia

Answer 70

In situ carcinoma can arise from surface epithelium which exhibits dysplasia. Dysplasia refers to the disordered growth and cytologic changes seen in epithelium, and dysplasia can (but does not always) progress to carcinoma in situ. Dysplasia often arises in metaplastic epithelium, but not all metaplastic epithelium is dysplastic.

Question 71

carcinoma in situ

Answer 71

Most benign neoplasms grow as cohesive, expansile (well circumscribed), masses, whereas malignant neoplasms often demonstrate progressive infiltration, invasion, and destruction of the surrounding tissue (there are exceptions). Invasive tumors such as carcinomas can elicit a fibrous stromal response called desmoplasia.

Some malignant epithelial neoplasms appear to evolve from a preinvasive, carcinoma in situ stage (e.g. carcinomas of the skin, breast, uterine cervix). Eventually the in situ carcinoma invades through the basement membrane, and the invasive tumor is now considered malignant because once stromal invasion is present, the carcinoma has the capability to metastasize.

In situ carcinoma can arise from surface epithelium which exhibits dysplasia. Dysplasia refers to the disordered growth and cytologic changes seen in epithelium, and dysplasia can (but does not always) progress to carcinoma in situ. Dysplasia often arises in metaplastic epithelium, but not all metaplastic epithelium is dysplastic.

summed up: in situ carcinoma have not yet broken through the basement membrane

Question 72

List and describe the six main categories of neoplasms.

Answer 72

Tumors of epithelial origin
Tumors of mesenchymal origin
Tumors of hematopoietic or lymphoid origin
Tumors of melanocytic origin
Tumors of the central nervous system (brain and spinal cord)
Tumors of germ cell origin

Question 73

List and describe the three pathways of metastatic spread.

Answer 73

Only malignant neoplasms can metastasize; benign neoplasms cannot (thus a neoplasm with metastases is malignant). Virtually all malignant neoplasms can metastasize, but some are more likely to do so than others. In general, the less differentiated, the more rapidly growing, and larger a malignant neoplasm is, the more likely it is to metastasize (of course there are exceptions).

Metastatic spread occurs through three pathways:

Direct seeding of body cavities or surfaces (e.g. seeding of peritoneal, pleural, pericardial, subarachnoid spaces)

Lymphatic spread: a very common pathway of spread of carcinomas, less common in sarcomas; the pattern of lymph node involvement follows the natural routes of lymphatic drainage. It is important to note that while worrisome for metastases, enlargement of lymph nodes in proximity to a malignancy could also be reactive.

Hematogenous spread: sarcomas exhibit this pattern of spread more commonly than lymphatic spread; carcinomas also exhibit hematogenous spread in addition to lymphatic spread; while the malignant tumor may seed the “next available capillary bed,” some carcinomas show preferential seeding of certain organs (e.g. lung carcinoma will preferentially metastasize to brain, bone, liver, adrenal).

Question 74

List the three most common cancers in men and women, and the one cancer responsible for most cancer deaths.

Answer 74

men: Prostate, lung, colon and rectum
women: breast, lung, colon and rectum
the most deaths: lung

Question 75

Discuss the relative role that aging, environment, and genetics play in the risk for developing cancer.

Answer 75

Genetic and environmental factors play a role in the pathogenesis of cancer, although environmental factors appear to be more significant in most common sporadic cancers.

Carcinogenic factors include exposure to UV light, work exposures, high-fat diet, alcohol abuse, cigarette smoke, exposure to human papillomavirus (HPV), etc., etc., etc. (just to name a few! – more on this later on)

Age has an influence on the likelihood of being afflicted with cancer. Most carcinomas occur in older individuals, with cancer being the primary cause of death among women aged 40 to 79 and men aged 60-79 (remember, when considering overall mortality, by all age groups, death by cardiovascular disease is #1, followed by cancer at #2). The rising incidence of cancer with age can be explained by the accumulation of somatic mutations and the decline in immune competence. In children the types of cancers that cause death are different than adults, and carcinomas are very rare. Instead, 60% of childhood cancer deaths are due to acute leukemias and primitive CNS malignancies.

Question 76

List the four types of genes typically mutated in cancer, and give examples of each, using common types of cancers as discussed in the lectures.

Answer 76

growth-promoting proto-oncogenes
growth-inhibiting tumor suppressor genes
genes that regulate programmed cell death (apoptosis)
genes involved in DNA repair

Question 77

Using only a few sentences for each, list the 8 essential alterations involved in malignant transformation of cells.

Answer 77

Self-sufficiency in growth signals (proliferate without external stimuli)
Insensitivity to growth-inhibitory signals
Evasion of apoptosis
Limitless replicative potential (avoid senescence)
Sustained angiogenesis (necessary for the tumor to grow)
Ability to invade and metastasize (tumor metastases are the cause of the vast majority of cancer deaths and depend on processes that are intrinsic to the cell or are initiated by signals from the tissue environment)
Defects in DNA repair (leads to genomic instability and mutations)
Escape from immune attack

Question 78

Describe examples of chemical carcinogenesis.

Answer 78

Chemical carcinogenesis occurs through a multistep process. Initiation results from exposure of cells to a sufficient dose of a carcinogenic agent, causing permanent DNA damage (mutation). Initiation is not sufficient for tumor generation; instead, additional exposure to a promotor is required. Promotors enhance the proliferation of the initiated (damaged) cells, contributing to the development of additional mutations.

Direct-acting agents require no metabolic conversion to become carcinogenic; indirect-acting agents require metabolic conversion of a procarcinogen to an ultimate carcinogen to become active. Other metabolic pathways may lead to the inactivation (detoxification) of the procarcinogen or its derivatives. Many of the known carcinogens are metabolized by cytochrome P-450-dependent mono-oxygenases. The genes that encode these enzymes are quite polymorphic, and individuals show great variability in the activity and inducibility of these enzymes. For example, cigarette smoke contains polycylic aromatic hydrocarbons which are metabolized to become active carcinogens by the product of the P-450 gene, CYP1A1. Light smokers with a highly inducible form of this enzyme have a sevenfold higher risk of lung cancer than smokers without this genotype.

Although any gene can be the target of a chemical carcinogen, commonly mutated oncogenes and suppressor genes, such a RAS and p53, are key targets.

In order for the unrepaired DNA damage to be heritable the initiated cell must replicate. The mitotic stimulus may come from the carcinogen itself, of from concurrent exposure to biologic agents, dietary factors, or hormonal influences that stimulate cell proliferation. These agents, which do not cause mutation but stimulate the division of mutated cells are known as promotors.

Question 79

Explain how the Warburg effect is used for cancer diagnosis .

Answer 79

Cancer cells quite consistently shift their glucose metabolism to aerobic glycolysis (Warburg Effect). This metabolic change can be used to visualize tumors via positron emission tomography (PET) scanning, in which patients are injected with 18F-flourodeoxyglucose, a non-metabolizable derivative of glucose that is preferentially taken up into tumor cells as well as normal, actively dividing tissues. The above PET scans show metastatic breast cancer in the liver and the right axilla (arrows).

Question 80

Define cachexia

Answer 80

Cachexia refers to the progressive loss of body fat and lean muscle mass along with weakness and anorexia that is associated with cancer. There is some correlation between the tumor burden and the degree of cachexia.

Affected individuals have an increased metabolic rate despite reduced food intake (in contrast to starvation).

Cachexia is not caused by the nutritional demands of the tumor. It is suspected that cachexia results from the action of soluble factors (such as tumor necrosis factor (TNF) produced by macrophages in response to the tumor or by the tumor cells, as well as other cytokines) that result in a catabolic state along with a suppressed appetite.

Unexplained weight loss can be a presenting symptom of cancer.

Question 81

Define grading and staging of malignant neoplasms in just a few sentences

Answer 81

Grading and staging of a malignant neoplasm is an attempt to quantify the probable aggressiveness and apparent extent and spread of the cancer in an individual patient.

Grading of a cancer is based on the degree of differentiation of the tumor cells, and in some cancers, the number of mitoses and/or architectural features as seen by light microscopy. Grade can be expressed numerically (1-3, 1-4) or descriptively (well differentiated, moderately differentiated, poorly differentiated).

Staging of cancers is based on the size and/or local degree of invasion, extent of spread to lymph nodes, and presence or absence of distant blood borne metastases. The major staging classification scheme used in the United States is the American Joint Committee on Cancer Staging (AJCC). AJCC staging uses the TNM classification – T for primary tumor, N for regional lymph nodes, and M for metastases.

In general, the staging of cancers is more useful clinically than the grade (there are exceptions, such as sarcomas).

Question 82

Describe what T,N,M refer to in the AJCC staging classification scheme.

Answer 82

T for primary tumor, N for regional lymph nodes, and M for metastases.

Question 83

Describe the first step in the pathologic diagnosis of neoplasms.

Answer 83

Obtaining a sample containing the tumor is the first step in pathologic diagnosis.

Types of tissue samples:
Lesion is biopsied with a scalpel, only a portion removed: incisional biopsy.
Lesion is excised with a scalpel, and thus removed: excisional biopsy.
Lesion is biopsied with a hollow core needle or punch (skin): needle or punch biopsy.
Mucosal based lesion is biopsied with appropriate device: mucosal biopsy (e.g. endoscopic mucosal biopsy of the stomach).
Cells of the lesion are aspirated with a fine needle and a syringe (known as fine needle aspiration) and smeared on a slide: cytology smear.
Cells from a fine needle aspiration (FNA) syringe are squirted into a small bottle of fixative, which is then processed like a tissue biopsy: cell block.

Blood or body fluid samples:
Drop of blood is smeared on a slide and stained: blood smear.
Bone marrow is aspirated and smeared on a slide and stained: bone marrow aspirate smear.
Bone marrow aspirate is allowed to clot, forming a small mass, which is then processed like a tissue specimen: clot section.
Bone marrow hollow core needle is used to obtain a core of tissue: bone marrow core needle biopsy.
Body fluid is aspirated, and the fluid is processed to place cells on a slide: cytology slide.
Body fluid is aspirated, and the fluid is spun down to produce a small pellet of cellular material, which is then processed as tissue: cell block.

Question 84

In just a few sentences, describe how an immunohistochemical stain works

Answer 84

Immunohistochemistry allows for the detection of cell products or surface markers on formalin fixed, paraffin embedded tissue. These immunohistochemical stains are used in the diagnosis or management of malignant neoplasms as follows:

Categorization of undifferentiated malignant tumors: (e.g. distinguishing an anaplastic carcinoma from high grade lymphoma or melanoma).

Determination of the site of origin of a tumor (e.g. is the lung adenocarcinoma really from the lung, or a metastasis from somewhere else).

Detection of molecules that have prognostic or therapeutic significance (e.g. presence of estrogen receptors, progesterone receptors, or overexpression of Her2/neu in breast carcinoma).

Question 85

Describe in just a few sentences the applications of molecular diagnostics with regard to cancer – i.e., how are these techniques used?

Answer 85

A variety of techniques are used and some applications are listed below:

Diagnosis of malignant neoplasms:
Some hematopoietic tumors are defined by their specific genetic abnormality (e.g. CML, APL), so detection of the abnormality is required for diagnosis. Certain sarcomas also have specific genetic abnormalities, so detection can help in definitive classification (e.g. Ewing sarcoma). It can sometimes be difficult to determine if a B lymphocyte or T lymphocyte process is monoclonal (neoplastic), so B cell immunoglobulin and T cell receptor gene rearrangement studies can be used. Sometimes gene expression profiles (using DNA microarrays) can be used to help type the cell or tissue of origin in high grade malignancies (e.g. adenocarcinoma of unknown origin).

Prognosis of malignant neoplasms:
Specific chromosomal abnormalities can be associated with a better or worse prognosis in certain malignancies (e.g. oliogodendrogliomas with loss of 1p and 19q only respond to therapy). Gene expression profiles are available for breast , prostate, and colon cancer and can help predict tumor behavior (prognosis) and guide therapy.

Detection of minimal residual disease (e.g., ALL, CML).

Diagnosis of a hereditary predisposition to cancer (e.g. BRCA1 or BRCA2 in breast cancer).

Question 86

Describe the common, major limitation of cancer biomarkers.

Answer 86

The concept behind tumor markers is that the malignant cells secrete a protein into blood or body fluids which can be measured, revealing the presence of tumor. Unfortunately, the biomarker is often secreted in both malignant and non-malignant conditions affecting the involved tissue. As such, the use of tumor markers cannot be used for definitive diagnosis of cancer, but in selected situations can assist in screening for cancer. One active area of research is proteomics, to look for more effective biomarkers of malignancy.

Tumor markers can be helpful for monitoring the effectiveness of cancer treatment as well as cancer relapse. Following successful initial treatment, the biomarker is no longer present. Return of the biomarker in the serum implies recurrence of disease.

Question 87

Define prevalence, sensitivity, specificity, and predictive value of both positive and negative tests.

Answer 87

Prevalence: percentage of individuals who have the disease in the population that is tested.
Sensitivity: percentage of individuals with the disease who have a positive test result.
Specificity: percentage of individuals without the disease who have a negative test result.

Predictive value of a positive test: percentage of individuals with a positive test result who truly have the disease: 160/7020 = .023 or 2.3% TP/(TP+FP)

Predictive value of a negative test: percentage of individuals with a negative test result who do not have the disease: 2940/2980 = .987 or 98.7% TN/(TN+FN)

Question 88

GENETIC PREDISPOSITION TO CANCER

Answer 88

Hereditary predispositions in addition to environmental factors do exist for cancers, including the common types. Thus an individual with a family history of cancer can have an increased risk. However, less than 10% of cancer patients have specific inherited mutations that predispose to cancer.

The genetic predispositions to cancer fall into three categories:

Autosomal dominant inherited cancer syndromes
Defective DNA-repair syndromes
Familial cancers (exact mutation(s) and mode of inheritance unclear)

Selected examples of these will be discussed later in this lecture, as well as in the specific organ system courses.

Question 89

NONHEREDITARY PREDISPOSING CONDITIONS FOR CANCER

Answer 89

Chronic inflammation, as can occur in a variety of conditions, can predispose to the development of cancer. A few examples are:

Ulcerative colitis: colonic adenocarcinoma
Helicobacter pylori gastritis: gastric adenocarcinoma, MALT lymphoma
Viral hepatitis: hepatocellular carcinoma
Osteomyelitis: carcinoma in the draining sinus
Hashiomoto’s thyroiditis: lymphoma

It is believed that the immune response may become “maladaptive” leading to tumorigenesis.

Specific entities (including “precancerous conditions”) will be discussed in the organ system courses.

Question 90

SELF-SUFFICIENCY IN GROWTH SIGNALS

Answer 90

Oncogenes are created by mutations in proto-oncogenes, resulting in oncoproteins, which resemble the normal products of proto-oncogenes except that the oncoproteins are often devoid of important regulatory elements, and their production in the transformed cells does not depend on growth factors or other external signals. This change promotes autonomous growth as a result of the “activating mutation.” Generally, only one allele needs to become mutant to create an effect.

Question 91

Insensitivity to growth-inhibitory signals

Answer 91

Tumor suppressor genes: products of these genes inhibit cell proliferation, preventing uncontrolled growth. Functions of tumor suppressor genes include:

Regulation of the cell cycle, particularly at the G1/S and G2/M checkpoints where the cell cycle is arrested in order to repair damaged DNA.
Regulation of nuclear transcription.
Regulation of cell differentiation, causing cells to enter a postmitotic, differentiated pool without replicative potential.

In contrast to oncogenes, both alleles for tumor suppressor genes need to be damaged for loss of growth inhibition (i.e., homozygous for the mutant allele, or, if one mutant allele is inherited, when the cell loses heterozygosity for the normal gene (known as loss of heterozygosity), although there are exceptions).

Expression of an oncogene in an otherwise normal cell leads to quiescence, or permanent cell cycle arrest (oncogene-induced senescence). Thus other defects (such as tumor suppressor gene mutations) are required for uncontrolled cell proliferation.

Question 92

Evasion of apoptosis

Answer 92

Apoptosis can occur through two pathways, an extrinsic death receptor pathway and a mitochondrial pathway. Defects can occur at several key points (numbered). One example involves BCL2 gene products which regulate and prevent apoptosis by limiting the release of cytochrome c. 85% of B-cell lymphomas of the follicular type carry a characteristic t(14;18) translocation which results in overexpression of the BCL2 protein. This protects the lymphocytes from apoptosis, allowing them to survive for long periods. Because BCL2-overexpressing lymphomas arise in large part from reduced cell death rather than explosive cell proliferation, they tend to be indolent (slow growing) compared with many other lymphomas.

Question 93

Limitless replicative potential (avoid senescence)

Answer 93

Most normal human cells have a capacity of 60-70 doublings, after which the cells lose their ability to divide and become senescent. This phenomenon is related to progressive shortening of the telomeres at the end of the chromosomes (telomeres prevent gene loss after multiple cell divisions, and short telomeres are recognized by the DNA-repair machinery as damaged DNA, leading to cell cycle arrest). Maintenance of telomeres is seen in virtually all types of malignant neoplasms, and in most cases, is due to the up-regulation of the enzyme telomerase (unlike malignant neoplasms, most benign neoplasms have normal telomerase activity).

Question 94

Sustained angiogenesis (necessary for the tumor to grow)

Answer 94

Solid tumors cannot enlarge beyond 1 to 2 mm in diameter unless they are vascularized. Cancer cells can stimulate neoangiogenesis, during which new vessels sprout from previously existing capillaries, or vasculogenesis, in which endothelial cells are recruited from the bone marrow. The tumor vasculature is abnormal; the newly formed vessels are leaky, dilated, and have haphazard connections. Angiogenesis is required not only for continued tumor growth but also for access to the vasculature and hence metastases. It is thought that angiogenesis is controlled by an increase in the production of angiogenic factors (such as VEGF) and/or a loss of angiogenic inhibitors. The factors may be produced by the tumor cells, by inflammatory cells (e.g. macrophages), or by stromal cells associated with the tumors. Numerous mediators and pathways have been proposed, and antiangiogenesis therapeutic agents have been created (e.g. monoclonal antibody to VEGF (vascular endothelial growth factor) .

Question 95

Ability to invade and metastasize (tumor metastases are the cause of the vast majority of cancer deaths and depend on processes that are intrinsic to the cell or are initiated by signals from the tissue environment)

Answer 95

Invasion and metastasis are the biologic hallmarks of malignant tumors and are the major cause of cancer-related morbidity and mortality. Remarkably, millions of tumor cells may be released into the circulation each day, but only a few metastases are produced, and some patients will never get metastases. Production of metastases involves two major steps:

Invasion of the extracellular matrix (ECM): this involves dissociation of tumor cells from one another, degradation of the basement membrane and interstitial connective tissue (or alternatively, ameboid migration through the basement membrane), attachment of the tumor cells to ECM proteins, migration of tumor cells within the ECM, and intravasation into blood vessels.

Vascular dissemination, homing of tumor cells, and colonization: once in the blood, tumor cells may attach to the endothelial cells of the first capillary bed available to the tumor; however, it is well known that certain tumors have preferential spread to certain organs. Such organ tropism may be related to different expression of endothelial adhesion ligands in the capillary beds of various organs, as well as different chemokine receptor expression in the various tumors. Colonization results from tumor cells secreting cytokines, growth factors, and ECM molecules that make the metastatic site habitable for the cancer cell (e.g. breast cancer metastatic to bone can activate osteoclasts, producing osteolytic lesions)

Question 96

Defects in DNA repair (leads to genomic instability and mutations)

Answer 96

Effective DNA repair mechanisms are essential to maintain the integrity of the genome. Individuals born with inherited defects in DNA repair mechanisms are at greatly increased risk of developing cancer. Many sporadic cancers also have defects in DNA repair mechanisms. DNA repair genes are not oncogenic, but their abnormalities allow mutations in other genes during the process of cell division.

Question 97

Escape from immune attack

Answer 97

Immune surveillance refers to the concept that one function of the immune system is to survey the body for malignant cells and destroy them. About 5% of individuals with congenital immunodeficiences develop cancers, about 200 times the rate of immunocompetent individuals.

Tumor cells can express a variety of antigens, some of which can be recognized by the immune system. Cell-mediated immunity is the dominant anti-tumor mechanism, and although antibodies can be made against tumors (e.g. anti-CD20 antibodies for treatment of B-cell lymphomas, a form of immunotherapy), there is no evidence that they play a protective role under physiologic conditions.

It is believed that tumor cells can develop mechanisms to escape or evade the immune system in immunocompetent hosts. Some proposed mechanisms are:

Selective outgrowth of antigen-negative variants.
Loss or reduced expression of MHC molecules.
Lack of costimulatory molecules (T-cell sensitization requires two signals, one by a foreign peptide presented by MHC molecules and another costimulatory molecule).
Immunosuppression (either as a result of therapy or by tumor products).
Antigen masking (surface antigens masked by glycocalyx molecules).
Apoptosis of cytotoxic T cells (tumor cells express proteins which induce T-lymphocyte apoptosis when they come into contact with the lymphocyte)

Question 98

growth-promoting proto-oncogenes

Answer 98

ABL-BCR fusion gene: results in increased tyrosine kinase activity (treat with tyrosine kinase inhibitor imatinib mesylate).
HER 2/neu: results in over expression of cell membrane epidermal growth factor receptor (in breast cancer, treat with monoclonal antibody to Her2/neu receptor, trastuzumab (Herceptin).
RAS: results in persistent activation of the RAS signal (KRAS for colon cancer; presence of KRAS in colon cancer can be predictive of lack of response to certain forms of chemotherapy).
BRAF: associated with melanomas.
KIT: results in activation of tyrosine kinase receptor c-KIT (occurs in gastrointestinal stromal tumors, treat with tyrosine kinase inhibitor imatinib mesylate).
Amplification of the N-MYC gene in neuroblastoma (above); chromosomal translocation and associated oncogenes in Burkitt lymphoma and chronic myelogenous leukemia (CML)

Question 99

growth-inhibiting tumor suppressor genes

Answer 99

TP53 (gene) is often listed as p53 (protein product of the gene); p53 acts as a “molecular policeman” that prevents the propagation of genetically damaged cells, through activation of temporary cell arrest (with subsequent DNA repair) , induction of permanent cell arrest (senescence), or programmed cell death (apoptosis). With loss of function of p53, DNA damage goes unrepaired and mutations accumulate in the dividing cells. Homozygous loss of p53 occurs in many cancers.
Women with strong family history of breast cancer can be screened for BRCA1/2 mutations.

Pathogenesis of retinoblastoma. Two mutations of the RB locus on chromosome 13q14 lead to neoplastic proliferation of the retinal cells. In the sporadic form both mutations at the RB locus are acquired by the retinal cells after birth. In the familial form, all somatic cells inherit one mutant RB gene from a carrier parent. The second mutation affects the RB locus in one of the retinal cells after birth.

Question 100

genes that regulate programmed cell death (apoptosis)

Answer 100

Apoptosis can occur through two pathways, an extrinsic death receptor pathway and a mitochondrial pathway. Defects can occur at several key points (numbered). One example involves BCL2 gene products which regulate and prevent apoptosis by limiting the release of cytochrome c. 85% of B-cell lymphomas of the follicular type carry a characteristic t(14;18) translocation which results in overexpression of the BCL2 protein. This protects the lymphocytes from apoptosis, allowing them to survive for long periods. Because BCL2-overexpressing lymphomas arise in large part from reduced cell death rather than explosive cell proliferation, they tend to be indolent (slow growing) compared with many other lymphomas.

Question 101

genes involved in DNA repair

Answer 101

Hereditary Nonpolyposis Colon Cancer Syndrome (HNPCC, autosomal dominant inheritance):

- Increased risk for carcinomas of the colon without previous polyps. 
- Results from inactivation of genes involved in DNA mismatch repair (affected individuals inherit one defective allele, and a second hit occurs in the colonic epithelial cell randomly, producing the defect – just like tumor suppressor genes).
- Characteristic finding in patients with mismatch repair defects is microsatellite instability (microsatellites are tandem repeats of 1-6 nucleotides found throughout the genome, in normal individuals the length of these microsatellites is constant).
- HNPCC accounts for only 2-4% of all colon cancers; microsatellite instability can be detected In 15% of sporadic colon cancers

Xeroderma pigmentosum: autosomal recessive inheritance, results in defective DNA repair, especially prone to develop skin cancer following exposure to UV light contained in sun rays.

Bloom syndrome, ataxia-telangeictasia, Fanconi anemia: all three disorders are autosomal recessive and are associated with chromosome instability; as such, there is hypersensitivity to DNA damaging agents such as ionizing radiation (Bloom syndrome and ataxia-telangiectasia) or chemotherapeutic agents (Fanconi anemia).

Question 102

Describe examples of radiation carcinogenesis.

Answer 102

Ionizing electromagnetic radiation (x-rays, gamma rays) as well as particulate radiation (alpha particles, beta particles, protons, neutrons) are known carcinogens . Increased incidence of cancers (initially leukemias, later solid tumors (e.g. breast, colon, thyroid, and lung) were seen following the WWII atomic bomb explosions. Increased incidence of malignancies have also been seen following nuclear accidents, and post-radiation sarcomas can rarely be seen following therapeutic radiation treatment.

UV rays (in particular, UVB light) from the sun is associated with increased risk of skin cancers (squamous cell carcinoma, basal cell carcinoma, melanoma). The carcinogenicity of UVB light is attributed to the formation of pyrimidine dimers in DNA, which are repaired by the nucleotide excision repair pathway. Patients with xeroderma pigmentosum have mutations which affect the function of this DNA repair pathway.

Question 103

paraneoplastic syndrome

Answer 103

Symptom complexes in cancer-bearing individuals that cannot be explained, either by the local or distant spread of tumor or by the elaboration of hormones indigenous to the tissue from which the tumor arose are known as paraneoplastic syndromes. They occur in about 10% of persons with the disease, and may represent the first clinical manifestation of an occult malignancy. They may cause significant clinical problems and can even be fatal.

Question 104

describe how flow cytometry works.

Answer 104

Flow cytometry is a technique that allows the quantification of cells in a stream of fluid by passing them by an electronic detection device. The cells can be incubated with fluorescent labeled antibodies which can bind to specific antigens on the cells. As the cells are detected, the fluorescent signal is also detected, allowing quantification of subpopulations expressing the antigen of interest. This technique is very useful in the identification and classification of leukemias and lymphomas. The above specimen shows a portion of a flow cytometry study on peripheral blood in a patient with chronic lymphocytic leukemia (CLL). The CD20 positive B-lymphocytes show kappa light chain restriction. Flow cytometry is performed on fresh tissue, blood, or body fluids.

Question 105

Compare the inheritance and penetrance of complex genetic disorders with diseases caused primarily by defects in a single gene.

Answer 105

 no simple Mendelian pattern of inheritance; affected individual share disease alleles and there are grades of expressivity and penetrance
 familial aggregation: relatives are more likely to develop a disorder
 environmental factors can play an important role
 complex diseases are more common among close relatives of the proband; greatest concordance is between monozygotic twins

Question 106

neoplasia

Answer 106

neoplasia is a disease process associated with uncontrolled cellular proliferation leading to a mass, or tumor (neoplasm)

Question 107

malignant

Answer 107

beyond control of tissue regulatory processes and capable of invading other tissues, which is called metastasis

Question 108

benign

Answer 108

tumors that do not spread are called benign, but they can become large, and cause health problems up to fatality

Question 109

metastatic

Answer 109

capable of invading other tissues

metastatic solid tumors are the most common aggressive cancer and are largely incurable

defined by the acquisition of genetic changes that permit invasion, evasion and translocation

!! recent research suggests that a subset of the human population carry a predisposition to metastasis

metastatic cells also have to overcome significant barriers

Question 110

carcinoma

Answer 110

epithelial: e.g., intestine, breast, lungs

Question 111

sarcoma

Answer 111

mesenchymal origin: bone muscle, connective tissue

Question 112

hematopoietic/lymphoid cancers

Answer 112

leukemias & lymphomas

Question 113

proto-oncogene

Answer 113

gain-of-function change for cancer

Question 114

oncogene

Answer 114

gene whose altered function or expression causes abnormal stimulation of cell division and proliferation; analogy of a broken car accelerator
oncogenes are dysregulated versions of endogenous genes called proto-oncogenes that are typically involved in normal growth stimulation, such as growth factors, receptor tyrosine kinases and transcription factors
 oncogenic mutations often turn “on” a stimulatory pathway and leave it stuck in the “on” position

Question 115

tumor suppressor

Answer 115

loss-of-function change required for cancer

Question 116

loss of heterozygosity

Answer 116

many cancers arise in cells that lose function of one allele, and then later lose function of the second wildtype allele; loss of this second allele is called loss-of-heterozygosity or commonly, LOH

Question 117

pharmacogenetics

Answer 117

a

Question 118

pharmacogenomics

Answer 118

a

Question 119

Discuss the implications of the clonal evolution hypothesis as it applies to the development, progression and clinical management of cancer

Answer 119

every tumor cell is equally capable of initiating neoplastic growth
genetic and epigenetic changes occur over time in individual cancer cells and that if such changes confer a selective advantage, they will allow individual clones of cells to outcompete other cells & expand
the new acquisition of genetic events underpins this model, but epigenetic and microenvironmental influences also likely play some role

Question 120

Discuss the implications of the stem cell hypothesis as it applies to the development, progression and clinical management of cancer

Answer 120

CSC hypothesis proposes that growth & progression of many cancers are driven by small subpopulations of CSCs
different types of cells in tumors: some cancerous, some stromal, some have different phenotypes
it has been observed for a long time that a large number of tumor cells were required to grow a tumor in a xenograft model in mice; if every cancer cell has the potential to form a tumor (clonal evolution hypothesis), then relatively few cells should be required, thus it may be that tumors contain a cell hierarchy in which a minority of SCs could self-renew & be able to regenerate a tumor
it has been assumed for a long time that cancer cells acquire new mutations that permit them to survive in their microenvironment (clonal evolution hypothesis), but it is also possible that this survival is a quality of a stem cell
only a very small fraction of cells within a tumor actually possess the ability to cause a cancer with plating efficiencies of 1/1000 to 1/5000
cancers are hierarchically arranged with CSCs lying at the apex

Question 121

Discuss the implications of the two hit hypothesis as it applies to the development, progression and clinical management of cancer

Answer 121

 almost all tumor suppressor genes act recessively at the cellular level this means that all function of the tumor suppressor gene must be lost
many cancers arise in cells that lose function of one allele, and then later lose function of the second wildtype allele; loss of this second allele is called loss-of-heterozygosity or commonly, LOH
most inherited cancers are considered autosomal dominant, not because a single mutant tumor suppressor gene causes the cancer but because the germline mutation is invariably followed by loss of the wildtype allele in a subset of cells
 Inherited tumor suppressor mutations are considered dominant at the level of the organism but recessive at the level of the cell

Question 122

Provide an example of how gene expression profiling of cancers can identify patient subpopulations with differential survival and responses to therapy.

Answer 122

classification of diffuse large B-cell lymphoma (DLBCL)
 DLBCL is the most common form of non-Hodgkins lymphoma, annual incidence of 25,000 cases
 clinically heterogeneous: 40% of patients respond to available chemotherapy, remaining 60% do not respond and die rapidly
explanation for the clinical differences is that DLBCL consists of two distinct cancers, with different genetic expression profiles
this was discovered by microarray analysis

 researchers created a “Lymphochip” consisting of genes known to be expressed in lymphoid cells
 analyzed a set of DLBCL in 42 patients and found that gene expression profiles could group the cancers into two classes based on their stage of differentiation; one set of cancers expressed genes associated with germinal center B-cells while the other set of cancers expressed genes associated with B-cells undergoing activation
 patients with “germinal center B-cell DLBCL” corresponded to patients with the cancers that responded to chemotherapy and where patients had good survival (76% 5-year survival)
patients with “activated B-cell like DLBCL” corresponded to patients with the cancers that failed chemotherapy and where patients had poor survival (16% 5-year survival)
initial study was confirmed in a different 240 patient study

Question 123

Provide examples of how genetic polymorphisms in the human population in genes involved in drug metabolism have a profound impact on drug toxicity and efficacy

Answer 123

a

Question 124

clinical implications of CSC hypothesis

Answer 124

cytotoxic agents are commonly designed to kill proliferating cells but are not curative because CSCs remain; thus the great majority of tumor cells, ie., the great bulk of tumors, arise from differentiation and do not possess tumorigenic potential on their own

normal SCs have innate multidrug resistance & tend to be resistant to chemotherapeutic agents and apoptosis, quiescent CSCs are also thought to be more resistant to chemo and other targeted therapies, and resistant to apoptosis, but differ from normal SCs by their impairment in differentiation
signaling pathways that regulate self-renewal of normal SCs (e.g., Wnt/beta catenin) become dysregulated in CSCs and lead to the expansion of the proliferative cell population, but current therapeutics do not target these pathways

effective strategies may include induction of differentiation of CSCs and drug targeting specific to CSCs, rather than the bulk of proliferating tumor cells

Question 125

Describe current treatment strategies for genetic diseases such as dietary modification

Answer 125

PKU, limit phenylalanine intake

Question 126

Describe current treatment strategies for genetic diseases such as avoidance,

Answer 126

lactose deficiency, don’t drink milk

Question 127

Describe current treatment strategies for genetic diseases such as modification of gene expression

Answer 127

 increase mRNA of either wildtype or mutant genes (if it still makes a functional protein)  induce expression of a different gene that can rescue the mutant phenotype, example: admininstration of butyrate to induce -globin gene (fetal hemoglobin) expression as a therapy for sickle cell anemia can repress expression of a dominant negative mutant using techniques such as RNAi; mutant Huntington mRNA would be a good target

Question 128

Describe current treatment strategies for genetic diseases such as protein replacement therapy

Answer 128

suboptimal strategy because the patient must come in every week
 can add a co-factor, e.g., pyroxidine/B6 for homocystinuria can replace the defective protein, e.g., 1-AT and Factor VIII (hemophilia) proteins problems: proteins have short half-life, immune response, viral contamination, insufficient suppliesGaucher Disease: a success story

most prevalent lysosomal storage disease: 1/450 Ashkenazi Jews; ~ 1/50,000 in the general population
autosomal recessive deficiency in the enzyme glucocerebrosidase that degrades the substrate glucocerebroside in the lysosome
disease is caused by glucocerebroside accumulation, particularly in the macrophages of reticuloendothelial system, leading to gross enlargement of the liver and spleen; in addition bone marrow is slowly replaced by lipid-laden macrophages (Gaucher cells) that ultimately compromise the production of erythrocytes and platelets resulting in anemia and thrombocytopenia
protein targeting for Gaucher disease can be effective because the CNS is not involved, abundant human enzyme is available from placenta and cultured cells, the only alternative is bone marrow transplantation (a high risk procedure), and the biology of macrophages is sufficiently understood
>2500 patients with Gaucher disease are now being treated with glucocerebrosidase enzyme replacement therapy; even though it is expensive and requires weekly infusions it has produced significant clinical benefits such as increases in platelets and hemoglobin levels and a decrease in the enlargement of spleen and liver
strategy depends on a modification of the carbohydrate that normally decorates the glycoprotein; terminal sugars are removed, permitting them to target macrophages, where they bind cell surface mannose receptors and are internalized and delivered to the lysosome
success is based on: 1) knowledge of the gene, protein and its function; 2) knowledge about disease pathogenesis; 3) knowledge about target macrophage biology

Question 129

Describe current treatment strategies for genetic diseases such as bone marrow transplants.

Answer 129

bone marrow transplantation
 effective treatment for some hematopoietic cancers and immune system disorders such as Severe Combined Immune deficiency (SCID, Fanconi’s anemia, lysosomal storage diseases, thalassemias, others
 used when the defect arises in bone marrow stem cells
 host tissue must not be irreversibly damaged
 can be used in combination with genetic engineering of a person’s own bone marrow stem cells
need a compatible donor (such as close relative)
problems with HSC transplants include significant mortality and morbidity from infection due to the required immunosuppression

cord blood
 placental cord blood is a rich source of HSC’s that has advantages over bone marrow in that recipients are more tolerant of histoincompatible placental blood than other allogenic donor cells, the wide availability of cord blood increases the number of donors, and risk of graft-versus-host disease is greatly reduced

Question 130

Describe gene therapy strategies, both ex vivo and in vivo,

Answer 130

 ex vivo: transfer of a gene outside the body or a stem cell, followed by introduction into the body; advantage: does not require an efficient means to enter a cell since a rare engineered cell can be selected for in cell culture; disadvantage: difficult & time consuming
 in vivo: direct injection into the body using a vector; advantage: quick & easy; disadvantages: many, including targeting proper cells, immune responses, safety

Question 131

Describe strategies for stem cell therapy using both somatic stem cells and embryonic stem cells, and their relative advantages and disadvantages

Answer 131

ES cells
 totipotent/pluripotent cells that can differentiate into any cell type in the body
 isolated from the inner cell mass of a blastocyst
 capable of unlimited replication in cell culture
 can be instructed to differentiate into any cell & tissue type in vitro (hematopoietic, cardiomyocytes, neurons, endothelial cells, adipocytes etc) and then introduced into an animal/human where they can function normally
 many applications already to fix damaged tissue: pancreatic islet cells, PD, cardiac cells
 problems: need to properly differentiate cells before introduction; ES cells can cause cancer; ethical & political
 however, great promise for rebuilding damaged tissue

somatic stem cells
 most adult organs contain stem cells populations (bone marrow, liver, heart, intestine, brain, muscle, skin) that can be isolated, and are capable of self-renewal and differentiation into other cell types under the proper conditions
 trials have shown that these adult stem cells are multipotent and can be used to repair damaged tissues in animals/humans and present less of a risk for cancer than ES cells
 of all the adult stem cells bone marrow-derived stem cells and umbilical cord blood stem cells show the greatest promise, they can differentiate into a variety of other stem cells and tissues

Question 132

three complications in treating monogenic disorders

Answer 132

 gene may not have been identified; and even when the gene(s) is known knowledge of the pathophysiological mechanism is not understood, e.g., despite years of study the mechanisms underlying elevated phenylalanine impairment of brain development & function are poorly understood
 fetal damage may occur prior to diagnosis and cause irreversible pathological changes
 most severe clinical phenotypes are less amenable to intervention

additional long term complications
 treatment is successful at first but subtle deficiencies appear later (e.g., PKU)
 treatment is successful in one tissue but problems develop later in other tissues (e.g., galactosemia, retinoblastoma)
 treatments initially show no harmful side effects but they develop later (e.g., infusion of clotting factor in hemophilia leads to severe immune response)

Question 133

effect of genetic heterogeneity on therapy

Answer 133

 proper treatment of a genetic defect requires knowledge of the specific defective gene, nature of its defect, genetic and biochemical pathways!
example of p53, which binds DNA as a tetramer - p53 mutations can act as a dominant negative thus if you develop a gene therapy that introduces wildtype p53 into a cancer cell that has a dominant negative p53 all of the new wildtype p53 may be “poisoned” by the mutant protein

Question 134

gene therapy

Answer 134

 definition: modification of cells to produce a therapeutic effect; based on recombinant DNA technology that permits the introduction of new genes and possibly the removal of damaged genes
 offers the hope of permanently reversing a genetic defect

Question 135

reasons to do gene therapy

Answer 135

 compensate for a mutant cellular gene with a loss-of-function mutation
 replace or inactivate a dominant mutant gene (e.g., HD, degrade mutant HTT gene)
 pharmacological effect (e.g., cancer)

Question 136

gene therapy guidelines

Answer 136

 defective gene & biochemistry has been described
 cDNA or functional version of the gene exists
 substantial disease burden & favorable risk-benefit
 confidence that the gene therapy will work based on knowledge of the defect
 reliable promoter to regulate gene expression
 good target cell
 data from model systems (eg., mouse) that gene, vector and delivery system all work
 protocol review by government

Question 137

the vectors employed to deliver the therapeutic gene

Answer 137

 vector is required to introduce a gene into a cell
 ideal vector should be safe, easily introduced into the target cell, and promote expression of the transferred gene for the life of the cell
 currently there is no perfect vector
 vectors can be classed as viral & non-viral
 viral vectors include: retroviruses, adenoviruses, adeno-associated viruses, herpesviruses
 non-viral vectors: naked DNA, liposomes, protein-DNA conjugates, artificial chromosomes

Question 138

retroviral vectors

Answer 138

 include onco-retroviruses and lentiviruses, can be made to enter virtually all target cells
 can be made simple & replication-defective and they are easy to engineer
 importantly, introduce DNA into the host genome
 can accommodate large transgenes
 problem with some retroviruses is that they require dividing cells to introduce DNA into the genome
 lentiviruses (such as HIV) can get around this problem and integrate DNA into quiescent cells
 problems? mainly safety

Question 139

adenoviral vectors

Answer 139

 advantages: can be generated at high titer, can infect a wide range of cell types, can accommodate very large genes (30-35 kb); some good examples in cancer gene therapy (p53, Rb)
 disadvantages: does not integrate into the genome so that expression is transient, causes a strong and sometimes damaging immune response, can result in toxicity in some normal cells

Question 140

adeno-associated vectors (AAV)

Answer 140

 advantages: similar to AV, no adverse effects in humans, can infect both dividing and non-dividing cells, while mostly episomal some can integrate into the host genome, examples include CF, Factor IX for hemophilia, muscular dystrophy, CNS diseases
disadvantages: until recently could only package small transgenes (5 KB) but recent improvements in vector design offer promise
AAVs are now the current preferred viral vector for clinical trials

Question 141

oncoretrovirus

Answer 141

infects proliferating cells, stably integrates into cell genome

advantages: permanent expression of transgene
disadvantages: effective only in proliferating cells, possibility of silenced transgene expression, insertional mutagenesis and oncogenesis

Question 142

lentivirus

Answer 142

broad tropism, stably integrates into cell genome

advantage: permanent expression of transgene
disadvantages: endogenous recombination with other retroviral sequences and reversion to HIV, insertional mutagenesis and oncogenesis

Question 143

herpes virus

Answer 143

central nervous system tropism

advantage: large packaging capacity
disadvantages: strong inflammation, neurotoxicity

Question 144

non-viral vectors

Answer 144

need-to-know: they just don’t work well

Summary:
 advantages (combined): lack biological risks associated with viral vectors; potential is unlimited
disadvantages: not been very successful; DNA tends to get degraded in lysosomes and not translocated to the nucleus, but research continues
Examples:
 naked DNA: cDNA & regulatory elements packaged in a plasmid
 liposomes: continuos lipid bilayer encompassing an aqueous solution containing plasmid DNA
 protein-DNA conjugates: DNA attached to a peptide that binds a receptor and is internalized
artificial chromosomes: minimal components of a natural chromosome combined with a therapeutic gene
nanoparticles

Question 145

siRNA in gene therapy

Answer 145

delivery of mutant gene or pathogen gene specific siRNAs is an active field of research
siRNAs can be designed to target a range of tissues, an example is lung, which can be targeted with either viral or non-viral vectors using delivery systems such as inhalers
examples of preclinical and clinical trials include knockdown of RNAs produced by the SARS virus in lungs, knockdown of mutant genes expressed by lung cancers and knockdown of genes that aggravate lung pathology in CF
knockdown of mutant Huntington mRNA is also being tested

Question 146

lysosomal storage diseases (LSDs)

Answer 146

70 distinct diseases, characterized by progressive storage of undigested or partially digested materials within the lysosome
caused by mutations in genes involved in lysosomal degradation (e.g., hydrolases)
can be managed by enzyme replacement therapy (ERT, see Gaucher Disease) and hematopoietic cell transplantation - but ERT cannot treat the neurological deficits present in ~ 75% of patients due to the blood brain barrier, plus the patients requires continual infusions
all are recessive diseases, with unaffected carriers, and low levels of wildtype protein (~ 1-20%) can significantly improve clinical phenotype
for that reason, gene therapy clinical trials underway (~20), AAVs and other vectors

Question 147

risks for gene therapy

Answer 147

 some deaths have occurred (adenoviruses, retroviruses); one famous case involved Jesse Gelsinger, a college student in PA who was in a clinical trial for OTC deficiency gene therapy using an adenoviral vector; he died of a severe immune response, causing the trial to be suspended; it was later determined that the head researcher at the University had a financial interest in the company that was financing the trial; Gelsinger should also have been excluded from the trial because he had health complications
 another famous example of X-SCID: first disease believed to be completely cured by gene therapy;one type of SCID is an X-linked disorder caused by defect in C cytokine receptor subunit; deficiency causes a block in T and NK cells growth & survival; previous treatments included BMT, but patients later developed additional problems; in trials patients’ BM cells were removed and infected with retroviral vectors carrying a wildtype C receptor gene; cells were reintroduced with dramatic clinical improvement.
problem: later SCID-treated children started developing fatal leukemias (a total of 5 out of 20 children) with at least two leukemias arising from retroviral insertion mutations into the LMO2 gene; the trials were suspended and the results significantly impeded the progress of human clinical trials
interestingly, similar trials for ADA-SCID were effective and safe