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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).
From Bardeesy et al., Semin Canc Bio, 2001Photos courtesy J. Glickman, Brigham and Women’s Hospital
Cancer is a genetic disease
Pancreatic adenocarcinoma
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
0 200 400 600 800 1000 1200
Incidence
Mortality
Thousands
LungBreastColo-rectalStomachLiverProstateCervix and uterusEsophagusBladderNon-Hodgkin’s lymphomaOral cavityLeukemiaPancreasOvaryKidney
Males Females
1200 1000 800 600 400 200
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 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.
RAS RAFProliferation /
Aggessive growth
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 OKBurkitt’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)
•Excellent prognosis
•Ewing’s sarcoma:•Surgical womp.•Different Hi-Dose chemotherapy•XRT post chemo
•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 (so-called ‘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.
12 months 0
PCR on the blood for BCR-ABL
• One problem: Low copy Bcr-Abl can be found in ‘normal’ people at modest frequency (carpe diem).
BMT 1 month 3 month 6 months 6.5 months++++ 0 0 + ++
DLI begun
Minimal Residual Disease
• 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.
BMT 1 month 3 months 6 months 6.5 months 12 monthsBcr-Abl ++++ 0 0 + ++ 0# CA cells 109 102 104 106 106.5 0
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.
• 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.
RNA expression profiling
X 40,000 spots per glass slide
From van de Vijver et al. NEJM 2002
Metastasis-free survival:
A company (Genomic Health) now sells molecular phenotyping as clinical service using this type of analysis.
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.
