Clinical Cancer Genetics

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Clinical Cancer Genetics

• Norman E. Sharpless, M.D.
• 966-1185
• NES_at_med.unc.edu

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Cancer Is

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

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

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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
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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).

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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)

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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.
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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.

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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!

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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.

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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.

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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.

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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

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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

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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.

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

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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.

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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.

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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).

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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
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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

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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

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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).

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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.

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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).

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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.

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Cytogenetics 2005 Chromosome painting and
Spectral karyotyping (SKY)
Specific Paints (DAPI counterstain)
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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.

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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.

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PCR on the blood for BCR-ABL

• One problem Low copy Bcr-Abl can be found in
  normal people at modest frequency (carpe diem).

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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.

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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

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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)

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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.

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

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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).

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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).

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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.
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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.

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X 40,000 spots per glass slide
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A company (Genomic Health) now sells molecular
phenotyping as clinical service using this type
of analysis.
From van de Vijver et al. NEJM 2002
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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.

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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.

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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).

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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.

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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.