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Clinical Cancer Genetics

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Cytogenetics: t(11;22) = Ewing s Specific Translocations: ... Desmin, Myf = rhabdomyosarcoma Cytogenetics The grand-mother of cancer genetic tests ... – PowerPoint PPT presentation

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Title: Clinical Cancer Genetics


1
Clinical Cancer Genetics
  • Norman E. Sharpless, M.D.
  • 966-1185
  • NES_at_med.unc.edu

2
Cancer Is
  • Inappropriate proliferation and resistance to
    differentiation and apoptosis.
  • Genomic instability.
  • Ability to grow where it ought not (i.e.
    metastasis).

3
Cancer is a genetic disease
Pancreatic adenocarcinoma
From Bardeesy et al., Semin Canc Bio, 2001 Photos
courtesy J. Glickman, Brigham and Womens Hospital
4
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

5
Estimated incidence and mortality WORLDWIDE, 2000
Males
Females
Lung
Breast
Colo-rectal
Stomach
Liver
Prostate
Cervix and uterus
Esophagus
Bladder
Non-Hodgkins lymphoma
Oral cavity
Leukemia
Pancreas
Ovary
Kidney
1200
1000
800
600
400
200
Thousands
Parkin et al., Eur Jour of Cancer 2001
6
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 lt
    85 y/o).

7
How do oncologists predict patient outcome?
  • Tumor type (i.e. histopathological
    characterization).
  • Tumor stage (TNMtumor, 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)

8
Lets Play a game
  • WHOS GONNA DIE??

I realize sounds callous, but we do this every
day in the clinic b/c we have to.
9
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.

10
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!

11
Case 0 Whos 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.

12
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.

13
Case 1 Whos 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-respons
    ive lymphoma.

14
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

15
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

16
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.

17
p53 status is often only of weak prognostic
value
Pancreatic cancer survival
18
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.

19
Comparing apples with apples
Proliferation / Aggessive growth
RAS
RAF
  • RAS mutations (15) not prognostic in melanoma
    (1993).
  • Almost all (gt80) melanoma has a RAS or RAF
    mutation.
  • But did we learn somethingyes, B-RAF inhibitors
    might make an excellent melanoma therapy.

20
If the obvious candidates dont work, what does?
  • Things that can be measured
  • Easily
  • Cheaply
  • Non-invasively
  • Reliably
  • Things that help discern dissimilar entities.
  • In most cases things that we identified
    empirically.

21
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).

22
What would you do when faced with sick child,
frightened parents, unsure pathologists (not to
mention zealous malpractice attorneys, etc.)?
Ewings sarcoma Surgery, chemo, XRTdo
OK Burkitts lymphoma Chemodo
great. Rhabdomyosarcoma Surgery, chemodo
so-so. Neuroblastoma Surgery, chemo, XRT (or
nothing)do so-so. First three are uniformly
fatal if not (or mis-) treated
23
How does one decide?
  • Cytogenetics
  • t(1122) Ewings
  • Specific Translocations
  • IgH-Myc Burkitts
  • Certain amplifications and deletions
  • N-myc neuroblastoma
  • Gene expression (by immunostaining)
  • Desmin, Myf rhabdomyosarcoma

24
What would you do
  • Burkitts lymphoma
  • Hi-Dose chemotherapy
  • Intrathecal chemotherapy (by serial lumbar
    puncture)
  • Excellent prognosis
  • Ewings sarcoma
  • Surgical womp.
  • Different Hi-Dose chemotherapy
  • XRT post chemo
  • Good prognosis

25
Cytogenetics
  • The grand-mother of cancer genetic tests
    (Philadelphia chromosome was identified as
    mini-chromosome in AML in 1960, t(922) 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 gt 1 month, often not that sensitive
    (many tumors dont grow in vitro).

26
Case 3 6 y/o with acute lymphoblastic leukemia
and tumor with
  1. Normal cytogenetics.
  2. Hyperdiploidy (too many chromsomes).
  3. 922 translocation (the Philadelphia
    chromosome).
  4. All are equally bad.

27
Cytogenetics are useful
  • t(922) 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 (so-called
    molecular CR).

28
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.

29
Cytogenetics 2005 Chromosome painting and
Spectral karyotyping (SKY)
Specific Paints (DAPI counterstain)
30
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
    (RNAremember, has to be easy to do).
  • Have begun to be used widely for assesing
    minimal residual disease.

31
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.

32
PCR on the blood for BCR-ABL
  • One problem Low copy Bcr-Abl can be found in
    normal people at modest frequency (carpe diem).

33
Minimal Residual Disease
BMT 1 month 3 months 6 months
6.5 months 12 months Bcr-Abl
0 0
0 CA cells 109
102 104 106
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.

34
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

35
Assays of gene expression
  • Currently, gt95 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)

36
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.

37
E ER/PR HHer2 BHer2/ER/PR negative
38
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)

39
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).

40
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).

41
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.
42
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 (gt10K).
  • Tumors are clustered by various statistical
    methods (unsupervised vs. supervised).
  • Hypothesis is that tumors in common clusters will
    behave in a clinically similar manner.

43
X 40,000 spots per glass slide
44
A company (Genomic Health) now sells molecular
phenotyping as clinical service using this type
of analysis.
From van de Vijver et al. NEJM 2002
45
Two Cautionary Tales
  • Medicine gtlt 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.

46
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.

47
Example II Autologous stem cell transplants in
breast cancer
  • In early 1990s, 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).

48
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.

49
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.
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