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Pathology
Neoplasia II, Molecular Neoplasia
09-23-08
Brian Bolerjack
toomanyminutos
1:
Continuation of last week, which was terminology and pictures. This time it’s molecular
neoplasia, how these things come about.
2:
The fundamental principles that you need to be familiar with. Tumors are monoclonal, resulting
from accumulation of nonlethal genetic damage. A single cell undergoes some mutagenic event, has
alteration of its genetic material, this is passed on to all offspring, and mutations accumulate. Targets of
genetic damage include Protooncogenes (growth promoting), Tumor suppressor genes (growth
inhibiting), Apoptosis regulating genes, Genes regulating repair. Carcinogenesis is a multistep process
with an accumulation of mutations.
3:
This is a cartoon from the book, and is of an isoform of an enzyme. The woman has two
versions of the enzyme, an A form and a B form. This could be of any informative enzyme that you have
two isoforms of. If we look at a neoplasm that arises, we would expect to see that all the cells in the
neoplasm are either one isoform or the other. The whole tumor arises from a single cell. You wouldn’t
expect to see a mixture of the isoforms. You don’t have polyclonal neoplasms.
4:
This is a schematic of what we are going through today. We have acquired DNA damage that
can be caused by many agents. These affect a normal cell, cause DNA damage. A repair mechanism
may be initiated, but if it fails the mutation is passed on and they accumulate. With accumulation
mutations you have progression of the tumor. So it’s a heterogeneous tumor even though it arises from
one stem cell. It acquires new mutations so the cells are variable within a tumor.
5:
How do tumors become malignant? It arises from self sufficiency in growth signals. Division of
the cells is not under the normal regulatory controls of cell division. Cells are insensitive to growth
inhibition. They can evade cell death, divide limitlessly (to an extent). They have to be able to sustain
angiogenesis, or blood vessel production, to provide the tumor with the nutrients it needs. They have to
have the ability to invade the basement membrane if it’s an epithelial tumor, to metastasize it must
invade the blood vessel, travel to a distant point, evade the immune system, implant wherever it goes,
and cause angiogenesis to provide nutrients. So it’s a complex system that has to happen for a tumor
to metastasize.
6:
Oncogenes. Oncogenes are the first genes we’ll talk about that drive neoplastic transformation.
Protooncogenes, oncogenes’ normal counterpart, are genes which function to promote normal growth
and differentiation. Their products drive the normal cell cycle. Oncogenes are derived from
protooncogenes with activation and loss of normal regulatory control. And so transcription and
production of oncoproteins is independent of growth factors or other external signals.
7:
There are lots of different oncogenes, this looks at the families of oncogenes. You can have
growth factors, growth factor receptors, signal transducing proteins, nuclear transcription activators,
and cell cycle regulators. And we’ll look at examples of these. Oncogenes may be activated by
overexpression, amplification, translocation, or point mutation. We will talk about examples of each of
those so we can talk about how this actually works.
Pathology
Brian Bolerjack
Neoplasia II, Molecular Neoplasia
toomanyminutos
09-23-08
8:
Some activation of oncogenes relies on a change in structure of the gene. This can happen
through translocation or point mutation. There can be changes in regulation of gene expression, so you
can have elevated levels of a normal protein.
9:
This is a cartoon from the book looking at RAS, which is membrane bound in the MAP kinase
pathway and is associated with a growth factor, and drives the cell cycle through activation of the MAP
kinase pathway. Growth factor binds to growth factor receptor, which is membrane protein, and you
have membrane bound RAS that is initially inactive binding GDP. With activation, GPD is released, GTP
binds with RAS activating it. Activated RAS activates the MAP kinase pathway, kinase is a
phosphorylation protein, and this activates transcription through the myc nuclear transcription factor.
With mutations in RAS, which is by a point mutation that alters the proteins structure, we block the
hydrolysis of GTP, so RAS stay in its activated form, continuing activation of the cell cycle unabaided.
Point mutations, or direct mutation of the protein, is one mechanism of oncogenes.
10:
Video of previous slide, skip.
11:
Another way oncogenes can be activated is by translocation. It can cause an increased
transcription of a normal gene product. This is Burkitt’s Lymphoma. We have a translocation from
chromosome 8 to chromosome 14. The myc oncogene, a nuclear transcription factor, is next to the IgG
gene, a very active area of transcription. You get increased myc production as a result. So increased
transcription secondary to a translocation.
12:
Translocation can also result in an altered protein. CML, where we have a translocation
between chromosome 9 and 22. The abl oncogene, a tyrosine kinase, is translocated to the breakpoint
cluster locus, and the two foci fuse to form a hybrid gene. It results in a tyrosine kinase that isn’t under
normal regulatory control. We have an oncogene activated by a translocation, free from regulatory
control.
13:
Oncogenic activation by amplification. Don’t remember the tumor, it’s a neural tumor. You
have amplification of myc just by increasing the number of copies of the gene. If you do a chromosomal
prep you can see there is an elongated band, known as a homogeneous staining region. You have
multiple copies of the gene within the chromosome, and you can also have transcriptionally active
extrachromosomal genetic material known as double minutes, and with increased copy numbers you
will have increased transcription of the gene.
14:
Another gene that is clinically important is HER2/neu which is important in breast cancer. This is
an example of amplification by increased copy numbers. With amplification of HER2/neu, patients did
worse. Initially this was reported as a poor indicator of breast cancer. But more recently a drug that
targets this membrane receptor has been developed and is called Receptin. We have a targeted therapy
for the oncogene if the patient expresses the specific oncogene. We are entering an age where you can
give specific drugs based on the antigenic expression of a tumor.
15:
This is a membrane bound protein, so we can look at it through Ab staining, immunoperoxidase
stain to see if there is overexpression. Overexpression of the membrane bound protein will highlight the
cytoplasmic membrane.
Pathology
Brian Bolerjack
Neoplasia II, Molecular Neoplasia
toomanyminutos
09-23-08
16:
You can also do direct probing by in situ hybridization to look at copy numbers. This is an
example of FISH the dots on the left are in a non-amplified cell. You wouldn’t expect to see more than
two copies of the gene. On the right there are multiple copies the HER2 gene.
17:
Tumor suppressor genes, known as antioncogenes, cancer suppressor genes, inhibit the cell
cycle. Important to remember: Knudsen’s two-hit hypothesis, which states that both alleles of the
tumor suppressor gene must be mutated for loss of inhibitory function. If one allele is mutated you will
have 50% of the product and that will usually be enough. You have to lose both alleles. The classic
example is the Retinoblastoma protein.
18:
This is a cartoon showing the normal function of the retinoblastoma protein. It serves as the
gatekeeper of the cell cycle for progression from G1 to S. In its phosphorylated or activated form, the
cell can progress from G1 to S and continue through the cell cycle.
19:
This is a little more complex. The phosphorylation allows release of the E2 transcription factor,
which allows transcription to occur. In its hypophosphorylated form E2F is bound and it blocks
transcription. Anything that activates would be in the oncogene category and anything that inhibits
would be in the tumor suppressor gene category.
20:
Retinoblastoma is a rare ocular tumor of childhood. It is a prototype of cancer cause by loss of
tumor suppressor genes. Most are sporadic, but 40% are familial born.
21:
Gross (tan fleshy masses) and histological (small blue cell tumor) pictures.
22:
In its sporadic form, all somatic cells have the normal retinoblastoma alleles. A mutation occurs
in a single allele, and at that point there is normal function. But if there is another mutation in that cell
or its offspring, you lose the inhibitory function of the gene, and you develop the tumor. In its familial
form, there is already a single allele mutation. All the somatic cells have one mutated allele. If there is a
single mutation in the other allele, then you will develop the tumor.
23:
TP53 (p53) is the single most common target for genetic alteration in human tumors. In LiFraumeni Syndrome, individuals are born with one defective p53 gene. This makes them susceptible to
many tumor types, mainly bone soft tissue sarcomas. P53 starts to apply the brakes on the cell cycle
and allow DNA repair.
24:
This is a cartoon from the book. In a normal cell you have DNA damage to some degree, and
when this happens p53 is activated and binds to the DNA. It upregulates certain target genes, cyclin
dependent kinase inhibitor, growth arrest and DNA damage 45 that is responsible for DNA repair,
upregulation of p21 results in G1 arrest. This gene is involved in repair, if there is repair you have a
normal cell. p53 also regulates the bax/bcl2 ratio. Bax drives apoptosis, and with failure of repair the
cell undergoes apoptosis. This is with normal p53 function. You have an attempt at repair, with failure
of repair you get apoptosis, so the damage can’t be passed to offspring. With a mutated p53, we can’t
put the brakes on the cell cycle, you have less repair, and you get mutations passed on to future cells, so
you have neoplastic transformation.
25:
Apoptosis is controlled cell death, important in embryogenesis and passing on mutations. Bcl-2
prevents apoptosis, bax promotes apoptosis. The regulation of the two is under p53.
Pathology
Neoplasia II, Molecular Neoplasia
09-23-08
26:
Brian Bolerjack
toomanyminutos
When the ration of bax to bcl2 is high, apoptosis occurs.
27:
The layering gets complex. You have cyclin dependent kinase feeding into retinoblastoma and
its formation. You also have cyclin kinase inhibitors that regulate the cyclin dependent kinases. You
don’t need to know all the details.
28:
Central ideas. Carcinogenesis is a multistep process requiring accumulation of multiple genetic
alterations. Molecular targets for carcinogenesis include protooncogenes, tumor suppressor genes,
genes regulating apoptosis, and genes involved in DNA repair. As a rule, cancers exhibit multiple genetic
alterations involving activation of several oncogenes and loss of two or more tumor suppressor genes.
29-30: A few more concepts in neoplasia we need to understand.
31:
Early in neoplastic transformation, we have a single cell that divides, so with each generation
you have a doubling of the tumor size. As the tumor grows, a number of cells drop into the
nonproliferative pool. Some will acquire lethal mutations, some will outrun their blood supply and
necrose. Some become senescent and drop out, could be nutritional. Some will undergo more
mutation and differentiation. Initially all the cells are growing and dividing. As the tumor grows, the
majority of tumors are not actively dividing.
That’s important because most chemo therapy is only effective against growing, dividing cells.
So most cells of a mature tumor will not be affected. Things are done to drive a larger percentage of
cells into the proliferative pool. Surgical debulking, you take away a lot of the mass and more cells will
move into the proliferative pool, and radiation debulks the tumor to drive the cells into the proliferative
phase. You kill off a large number and drive the remainder into the proliferative pool so the therapy is
more effective.
32:
Tumor progression. Subclones are generated from the original malignant cells. The most fit
survive and predominate in the tumor. The rate at which subclones with additional phenotypes develop
is variable.
33:
A normal cell undergoes neoplastic transformation, becomes a malignant cell. This tumor
requires fewer growth factors, which would be a survival advantage. So the tumor begins to grow faster
without requiring as many nutrients. Some will undergo nonviable mutation so there are no offspring
produced. Additional features may be acquired, the ability to evade the immune system, invade. We
have a heterogeneous tumor with an accumulation of mutations, which will provide some advantage
which will allow it to metastasize.
34:
This is very complex, and you think how could it ever happen. The tumor is able to invade
through the basement membrane and blood vessel, it evades the immune system, travels to another
site, extravasates through the blood vessel, sets up, and induces angiogenesis so it has a blood supply.
The lesson is that mutations occurring all the time. Some of the early mutations in neoplastic
transformation sets you up to be able to have more mutations. Once you have a p53 mutation, you are
more likely to pass on mutations because you no longer have the ability to regulate when cells should
apoptose.
Pathology
Brian Bolerjack
Neoplasia II, Molecular Neoplasia
toomanyminutos
09-23-08
35:
Why do we get mutations? Carcinogens! There are chemical agents, direct (directly affect the
DNA) or indirect (must undergo some metabolic transformation before they are carcinogenic) acting.
They can be inhaled toxins like in cigarette smoke, things we put in our bellies or things we put on our
skin. The ras gene is commonly affected by chemical carcinogens (dietary).
36:
There are viral carcinogens. We have RNA viruses, HTLV-1 which is associated with leukemia.
Hepatitis C virus is associated with hepatocellular carcinoma. DNA viruses, human papillomavirus is
associated with cervical cancer, certain oropharyngeal squamous cell carcinomas, particularly tonsilar
carcinomas. Epstein-Barr virus with nasopharyngeal carcinomas. Hep B virus with hepatocellular
carcinoma.
37:
Radiation can be a carcinogen by causing cross linking of DNA. UV from sunlight is associated
with skin cancer: squamous cell carcinoma, melanomas, basal cell carcinomas are associated with sun
exposure. Ionizing and nuclear radiation increased rates of leukemia and tumors in Hiroshima and
Nagasaki. Chernobyl children have increased rates of papillary thyroid carcinoma. There is a long latent
period because there must be an accumulation of molecular events in order to get neoplastic
transformation.
38:
Colorectal carcinoma serves as a model to explore the progression of tumors. It has well
defined clinical progression and correlating histology. First, you have normal mucosal surface and crypt
epithelium. You uniform crypts lined by goblet cells with mucus toward the lumen of the crypt and
nuclei basally oriented. The earliest histological sign you can see is aberrant crypt foci (histologic, no
clinical correlation). We see these in patients with other lesions. You see decreased mucus production
and stratification of nuclei. They are starting to go to different levels throughout the cell.
Then we develop an adenomatous polyp, a clinical lesion that can be seen. A polyp is a raised
lesion from an epithelial surface. It can be attached broad (sessile) or it can be pedunculated on a stalk.
This is the earliest clinical sign and this is why older people have colonoscopies. You can determine risk
of cancer and small polyps can be removed at the time of colonoscopy. Then you have invasive
carcinoma and can develop the ability to metastasize.
39:
What do you see here? This is longitudinally opened colon. There is a little lesion on a stalk
with a dark brown cap. It is an adenomatous polyp. A pedunculated polyp in the colon.
40:
You can see two polyps here. Normal colonic epithelium.
41:
Histologically, you see normal epithelium, and it is fairly clear there because of abundant mucus
content. If you look in the cap of the stalk of the polyp it become much darker because you have
decreased mucin production and you have the nuclei moving throughout the cells.
42:
High magnification, nuclei are sitting toward the lumen, and should not be there. So these are
the characteristics of an adenoma.
43:
This is a large lesion in the colon, maybe 3cm. Can’t tell if it is malignant or not grossly. It could
be a large sessily adenoma, or an adenocarcinoma. Must look histologically.
44:
Another large lesion with central ulceration, so it is almost certainly an invasive
adenocarcinoma.
Pathology
Neoplasia II, Molecular Neoplasia
09-23-08
Brian Bolerjack
toomanyminutos
45:
Here is a cross section of a carcinoma and you can see invasion through the muscularis propria
out into the soft tissues, probably have lymph node metastasis.
46:
Histologically you would see this. You know it’s an adenocarcinoma, a malignant neoplasm of
epithelial tissue of glandular origin, because you see glandular differentiation. It’s infiltrating. And there
is a bit of atypia, decreased mucin production and distributed nuclei.
47:
We can look at molecular events at genes at each step and see how they are affected. We have
talked about ras and that it undergoes activating point mutations.
48:
If we look at colorectal carcinomas, it seems to drive progression. Specific point mutations are
identified in 50% of colorectal adenomas larger than 1 cm. ras mutations are rarely observed in smaller
adenomas. These findings suggest that ras mutations are involved in adenoma progression.
49:
We will start a scheme with all the steps we looked at, from normal colonic epithelium to
metastatic carcinoma, and we have ras mutation between early and intermediate adenoma.
50:
p53 and its role as a tumor suppressor gene. Tumor suppressor gene on chromosome 17p13.1
Among the most common targets for genetic mutation in human cancers (Li-Fraumeni syndrome). p53
normally functions to inhibit cell cycle activity in response to DNA damage and to regulate apoptosis
(bax/bcl-2).
51:
17p losses and mutations are observed in 75% of invasive colorectal carcinomas. These changes
at the p53 locus are rarely observed in adenomas. Inactivation of p53 appears to be a late event in the
development of colorectal carcinoma.
52:
So this is between the progression for a late adenoma to a carcinoma, so the ability to invade.
53:
Deleted in Colon Cancer gene is located on 18q. The protein product of this gene acts as a cell
surface adhesion/receptor molecule. Loss of DCC expression is seen in over 70% of colorectal
carcinomas and half of large adenomas suggesting role as a tumor suppressor gene. Loss is rarely seen
in small adenomas.
54:
There are other genes in this area, but there is something in 18q21 that is acting as a tumor
suppressor gene so deletions are common.
55:
We can put that in progresson of adenomas.
56:
The APC (adenomatous polyposis coli) gene is located on chromosome 5q. Inherited genetic
mutation is associated with the familial polyposis coli syndrome. In this disease at childhood there will
be thousands of polyps that carpet the colon, and maybe the small intestine. There is 100% leading to
carcinoma if not treated. It is treated by total colectomy. The APC protein is a transcription regulator
through interactions with beta-catenin.
57:
This is an example. There is no normal colonic mucosa here. It is all adenomatous polyps.
Pathology
Brian Bolerjack
Neoplasia II, Molecular Neoplasia
toomanyminutos
09-23-08
58:
We find APC gene mutation in over 80% of sporadic colorectal adenocarcinomas. The mutation
rate in adenomas is similar to that in carcinomas. Mutations of the APC gene have been identified in the
earliest histologically defined lesions (ACF). These findings suggest that APC mutation is an early,
perhaps initiating event in colorectal carcinoma.
59:
So we can add that in the transition from normal colonic epithelium to the aberrant crypt foci.
60:
There are many other genes that can be implicated. We continue to acquire mutations that lead
to progression of the tumor. Sporadic cases of myc, myb, NEU, and cyclin amplification have been
identified in colorectal carcinoma. These oncogenes probably contribute to tumor progression,
particularly at late stages. Tumors are monoclonal but genetically heterogeneous.
61:
Those are added into the schematic there.
62:
We have loss of DNA methylation of posttranscriptional modification, which appears to be early
in adenoma formation.
63:
We are left with this cartoon from the book that is a little simplified. We have normal colonic
epithelium, homozygous loss of the APC locus on 5q (the cells don’t look normal, this happens in
aberrant crypt foci). This is a tumor suppressor gene so you must lose both alleles. You have a mutation
on ras of 12p that leads to formation adenomas. Homozygous loss of dcc or something on 18q, another
tumor suppressor gene leads to increase in adenoma size. Other mutations, loss of p53, leads to
invasive carcinoma and metastasis. Does it have to happen in this order? No. There’s no way to predict
it. This is a general scheme based on statistics. You could start with the p53 mutation and it go from
there. Tumors are monoclonal but heterogeneous. Tumor progression is from accumulation of
mutations that allows invasion of blood vessels, metastasis, and angiogenesis.