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Clinical Cancer Genetics
Norman E. Sharpless, M.D.
966-1185
[email protected]
Cancer Is:
• Inappropriate proliferation and
resistance to differentiation and
apoptosis.
• Genomic instability.
• Ability to grow where it ought not (i.e.
metastasis).
Cancer is a genetic disease
Pancreatic adenocarcinoma
From Bardeesy et al., Semin Canc Bio, 2001
Photos courtesy J. Glickman, Brigham and Women’s Hospital
Helpful Definitions:
• Leukemia: Malignant cells circulating in the
blood and bone marrow.
• Lymphoma: Malignant lymphoid cells in the
lymph nodes.
• Dysplasia: A “premalignant” condition of
almost any tissue characterized by an
abnormal histopathological appearance.
• Sarcoma: Mesenchymal tumor
• Carcinoma: Epithelial tumor
Estimated incidence and mortality WORLDWIDE, 2000
Males
Females
Lung
Breast
Colo-rectal
Stomach
Liver
Prostate
Cervix and uterus
Esophagus
Bladder
Non-Hodgkin’s lymphoma
Oral cavity
Leukemia
Pancreas
Ovary
Kidney
1200 1000
800
600
Incidence
Mortality
400
200
0
200
400
600
800
1000 1200
Thousands
Parkin et al., Eur Jour of Cancer 2001
Principles of neoplasia that can be
deduced from the epidemiology:
1. Certain tumors are more lethal than others, and
most cancers are lethal .
2. Certain tumors are associated with
environmental exposures (hepatitis B,
helicobacter,etc).
3. Smoking is really bad for you.
4. Considering the aging demographics, cancer
will be an even bigger problem in future (in fact,
CA already leading cause of death in US for
people < 85 y/o).
How do oncologists predict patient
outcome?
• Tumor type (i.e. histopathological
characterization).
• Tumor stage (TNM=tumor, node, metastasis).
• Tumor grade and degree of differentiation (i.e.
how much does it look like the tissue of origin).
• The patient (age, comorbid illness, “performance
status”)
Let’s Play a game:
WHO’S GONNA DIE??
I realize sounds callous, but we do this
every day in the clinic b/c we have to.
Predicting diagnosis and prognosis
are important:
• Helps tailor therapy (e.g. small cell lung
cancer vs. non-small cell lung cancer).
• Helps tailor therapeutic intensity (e.g.
acute leukemia)
• Helps guide follow-up in patients who
are NED (we never say “cure”).
• Helps patients live their lives.
Important medical concept:
“The fight does not always go to the
strong, and the race does not always go
to the swift…..
but that is how you should bet!”
Case 0: Who’s gonna die first?
1. A 44 y/o lady with met. breast cancer to
bone and liver.
2. A 51 y/o lady with breast cancer
metastatic to regional lymph nodes.
3. A 57 y/o lady with large cancer confined to
the breast, but invading the chest wall.
4. A 60 y/o lady with small cancer only in the
breast itself.
Stage is important:
• Stage refers to how advanced a cancer is.
• Stage correlates with two things: amount
of cancer cells and their propensity to
spread.
• For a given tumor type, advanced stage is
always worse than early stage.
• Not true across tumor types: Late stage
lymphoma better than early stage
pancreatic cancer.
Case 1: Who’s gonna die first?
1. A 44 y/o lady with met. breast cancer to
bone who is independent, exercises daily.
2. A 51 y/o with Stage III (advanced) chronic
lymphocytic leukemia who works full-time
and plays golf 1x/week.
3. A 27 y/o with HIV+ male with ESRD, hep.
C cirrhosis and good prognosis,
chemotherapy-responsive lymphoma.
What is performance status?
• ECOG Performance score
–
–
–
–
–
0 = fully active
1 = some symptoms
2 = Needs some assistance
3 = Needs complete assistance
4 = Near death
• PS is the MOST IMPORTANT PREDICTOR
OF LONG-TERM OUTCOME
The importance of performance status
• Careers have been made by only enrolling
the best patients in your clinical trial
• This can be very tricky and subtle:
– We only treat patients who can travel to the
NCI.
– We only operate on patients who respond to
chemotherapy before their surgery.
– We only analyzed the patients who received
full dose therapy.
– We only treated patients who complete the
Boston marathon…
Case 2: 44 y/o with advanced
pancreatic cancer:
1. Whose tumor has p53 deleted.
2. Whose tumor has p53 point
mutation.
3. Whose tumor has normal p53.
4. All are equally bad.
p53 status is often only of weak
prognostic value:
Pancreatic cancer survival
Why are the fundamental lesions of
cancer not so good at prognosticating?
1. Technical details (e.g. p53 is hard to
measure, multiple non-equivalent lesions,
etc).
2. To be of clinical value, a prognostic
variable has to be really easy to
determine, cheap, reproducible, etc.
3. Most interesting scientifically: these
lesions are the sine qua non of the
cancers themselves.
Comparing apples with apples:
RAS
RAF
Proliferation /
Aggessive growth
• RAS mutations (15%) not prognostic in
melanoma (1993).
• Almost all (>80%) melanoma has a RAS
or RAF mutation.
• But did we learn something—yes, B-RAF
inhibitors might make an excellent
melanoma therapy.
If the obvious candidates don’t work,
what does?
1. Things that can be measured:
•
•
•
•
Easily
Cheaply
Non-invasively
Reliably
2. Things that help discern dissimilar entities.
3. In most cases: things that we identified
empirically.
The “small round blue cell tumor”
• Classic diagnostic dilemma: poorly
differentiated, rapidly growing tumors of
small children.
• Tumor site, age of child, certain blood
tests helpful (but none are perfect).
• Treatments and prognosis are totally
different (and it could be one of four
things).
What would you do when faced with sick child,
frightened parents, unsure pathologists (not to
mention zealous malpractice attorneys, etc.)?
Ewing’s sarcoma: Surgery, chemo, XRT—do OK
Burkitt’s lymphoma: Chemo—do great.
Rhabdomyosarcoma: Surgery, chemo—do so-so.
Neuroblastoma: Surgery, chemo, XRT (or nothing)—do so-so.
First three are uniformly fatal if not (or mis-) treated
How does one decide?
• Cytogenetics:
– t(11;22) = Ewing’s
• Specific Translocations:
– IgH-Myc = Burkitt’s
• Certain amplifications and deletions
– N-myc = neuroblastoma
• Gene expression (by immunostaining)
– Desmin, Myf = rhabdomyosarcoma
What would you do…
•Burkitt’s lymphoma:
•Hi-Dose chemotherapy
•Intrathecal chemotherapy
(by serial lumbar
puncture)
•Ewing’s sarcoma:
•Surgical womp.
•Different Hi-Dose
chemotherapy
•XRT post chemo
•Excellent prognosis
•Good prognosis
Cytogenetics
• The grand-mother of cancer genetic tests
(Philadelphia chromosome was identified as
“mini-chromosome” in AML in 1960, = t(9;22)
in 1973).
• Done by culturing tumor cells, arresting them
in mitosis, and making metaphase spreads.
• Chromosomes are stained and interpreted by
a cytogeneticist.
• Takes days to > 1 month, often not that
sensitive (many tumors don’t grow in vitro).
Case 3: 6 y/o with acute
lymphoblastic leukemia and
tumor with:
1. Normal cytogenetics.
2. Hyperdiploidy (too many
chromsomes).
3. 9;22 translocation (the
‘Philadelphia chromosome’).
4. All are equally bad.
Cytogenetics are useful:
• t(9;22) makes bcr-abl
fusion protein.
• Correlates with bad
prognosis in ALL.
• Molecular target of
Gleevec (and predicts
Gleevec response).
• Can be followed as
marker of response (socalled ‘molecular CR’).
Pediatric Acute Lymphoid
Leukemia, 5-year survival
rates:
>50 chromosomes
>90%
40-50 chromosome ~80%
Ph+
<30%
Ph+ ALL gets an up-front BMT,
other kids get a trial of
chemotherapy
Cytogenetics and Prognosis
• Can signify prognosis that is:
– Good: iso12p in mediastinal “carcinoma of unknown
primary” = germ-cell tumor
– Average: 46XX (i.e. normal) in AML
– Bad:
Ph+ in ALL; 7q- in AML
Complex karyotype in solid tumors
• The oncologists’ easy to recall rule to
cytogenetics: if the report goes more than one
page, the prognosis is bad!.
• Deep Observation: pediatric cancers tend to
have simple cytogenetics, while adult cancers
are more complex.
Cytogenetics 2005: Chromosome painting
and Spectral karyotyping (SKY)
Specific Paints
(DAPI counterstain)
SKY
Visible
Enhanced
Chromosomal Translocations
• Replacing cytogenetics in many areas
(EWS-FLI, BCR-ABL, etc.) when the
target lesion IS KNOWN.
• Usually identified by PCR (DNA), rarely
RT-PCR (RNA…remember, has to be
easy to do).
• Have begun to be used widely for
assesing ‘minimal residual disease.’
Minimal Residual Disease
• 42 year old man with very high white blood
cell count, anemia, high platelets.
• Smear shows lots of well-differentiated
myelocytes (WBCs), some basophils.
• CML (chronic myelogenous leukemia, always
BCR-ABL positive).
• Treated with chemotherapy, total body
irradiation, and BMT.
• Cytogenetic remission in bone marrow at 6
months post-BMT.
PCR on the blood for BCR-ABL
BMT 1 month
++++
0
3 month 6 months 6.5 months 12 months
0
+
++
0
DLI begun
• One problem: Low copy Bcr-Abl can be
found in ‘normal’ people at modest
frequency (carpe diem).
Minimal Residual Disease
Bcr-Abl
# CA cells
BMT 1 month
++++
0
109
102
3 months
0
104
6 months 6.5 months 12 months
+
++
0
106
106.5
0
• Probably 2-4 logs more sensitive than cytogenetics.
• Affords the opportunity to treat small numbers of tumor cells
that are clinically silent, but the cause of relapse
(‘consolidation’).
• Consolidation can be good old-fashioned chemotherapy
(HiDAC, stem cell transplants etc), much interest in novel
therapies (immunotherapy, monoclonal antibodies, etc) in
this setting.
Other genetic events:
• Now have tests beyond cytogenetic
analysis used clinically for amplifications
(too much of a gene), deletions (too little of
a gene).
• Adult carcinomas characterized by
wholesale gains and losses.
• Mostly of scientific interest now.
• Examples: N-myc copy # important in
neuroblastoma, 13q deletion adverse in
myeloma
Assays of gene expression
• Currently, >95% is immunohistochemistry,
ELISA or flow cytometry (that is, antibodies
are used to stain the tumor).
• RNA methods are generally too unreliable for
widespread clinical use.
• RT-PCR is done in a few specific
circumstances (e.g. tyrosinase expression to
rule-in amelanotic melanoma)
Case 4: Who is gonna to live longest?
64 year old with unresectable breast
cancer whose tumor:
1. Expresses the estrogen and progesterone
receptors (ER/PR+).
2. Expresses Her2, the target of Herceptin
(HER2+).
3. Does not express any of these (‘triple
negative’)
4. All are equally bad.
E = ER/PR+
H=Her2+
B=Her2/ER/PR negative
IHC / ELISA / Flow
• Conjugated antibodies
bind cognate antigen
(e.g. CD3 on T-cells,
estrogen receptor on
mammary cell)
• Ab binding detected
by fluorescence or
chemical reaction (e.g.
horseradish
peroxidase)
An interesting observation
about gene expression tests:
• Usually, we measure genes that are
pathologically unimportant (CD3, vimentin,
keratins etc); to help determine tumor type.
• We are beginning, however, to have tests for
pathogenic molecules (e.g. Estrogen receptor).
• Even better, some of these molecules are good
targets for biologic therapy (e.g. anti-CD20 =
Rituxan, anti-HER2 = herceptin).
Enzyme assays
• Although not generally thought of as ‘cancer
genetics’; tumor enzyme assays are the
oldest clinically useful tests of gene
expression.
• In the old days, all leukemia was typed based
on enzymatic profiles, and myeloperoxidase
(MPO) is still used to tell AML from ALL
(although now can be done using an antibody
to MPO).
Cancer Genetics: the future
RNA expression profiling on
oligonucleotide microarrays
is capable of measuring the
expression of thousands of
genes in a tumor
simultaneously.
Based on expression, one
can “cluster” like tumors and
optimize therapy.
RNA expression profiling
• mRNA from tumor is converted to DNA and
labeled; then hybridized to array.
• array is of oligonucleotides or complementary
DNAs (several versions of arrays at present).
• Arrays represent large numbers of genes
(>10K).
• Tumors are clustered by various statistical
methods (“unsupervised” vs. “supervised”).
• Hypothesis is that tumors in common clusters
will behave in a clinically similar manner.
X 40,000 spots per glass slide
Metastasis-free survival:
A company (Genomic
Health) now sells
molecular phenotyping as
clinical service using this
type of analysis.
From van de Vijver et al. NEJM 2002
Two Cautionary Tales:
• Medicine >< Science.
• Medicine (appropriately) is very
conservative and moves much more slowly
than science.
• Blinded, randomized trials are required to
change the standard of care (cost millions,
require years of follow-up).
• Pathologists will be doing IHC and
metaphase spreads 10 years from now.
Example I: Prostate Specific Antigen
• PSA identified as marker of prostate cancer in
1980.
• It is, far and away, the best “tumor marker.”
• Still, who, if anyone, should have screening
PSA?
• What should you do in 70 y/o with high PSA? In
an 80 y/o?
• Clearly PSA has been a boon to radiation
oncologists and urologic surgeons, but still very
unclear if elderly men with indolent cancer
benefit from treatment.
Example II: Autologous stem cell
transplants in breast cancer
• In early 1990’s, several non-randomized trials (Phase
II) demonstrated impressive responses to high-dose
chemotherapy in breast cancer.
• Doses of chemo were so high, to survive patients
required reinfusion of their own hematopoetic stem cells
after chemo (so-called ‘stem-cell transplant’).
• In 1995, Bezwoda et al. reported a 90 patient study with
51% complete remission rate in metastatic breast
cancer with high-dose therapy and stem cell rescue (vs.
5% CR rate in women treated conventionally).
Example II: Autologous stem cell
transplants in breast cancer (cont.)
• Every large CA center in USA (and several small
ones too) began offering ASCT.
• Despite widespread physician skepticism and vastly
increased cost and toxicity, thousands of women
were treated in this way.
• 2001: Three large randomized trials showed no
benefit of ASCT.
• 2001: Bezwoda article was retracted after auditors
concluded the results had been FABRICATED.
Clinical Cancer Genetics
• Cancer is a genetic disease.
• Prognosis and therapy are based on tumor type,
stage and grade; and patient characteristics.
• Clinical cancer genetics, in 2005, is comprised of
cytogenetics, detection of chromosomal
translocations and amps/dels, and limited assays
of gene expression (IHC, ELISA, flow).
• We use these for diagnosis, therapy,
prognostication, and assessment of minimal
residual dz.
• New technology is exciting, but we have to be
careful.