Clinical Cancer Genetics

1. Clinical Cancer Genetics • Our understanding of the genetics of cancer is beginning to help us design better therapies. • This same understanding, however, has long (>30 years) been helping us make decisions about diagnosis and prognosis. • This is getting better all the time (we’ll talk about why).

2. 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 (no evidence of disease, we never say “cure”). • Helps patients live their lives.

3. Cancer Is: • Inappropriate proliferation and resistance to differentiation and apoptosis (Rb, p53, PTEN). • Genomic instability (p53, MIN vs. CIN,short telomeres?, others). • Ability to grow where it ought not (i.e. metastasis—RAS?, molecular mechanisms not clear).

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

5. Why are the fundamental lesions of cancer not so good at prognosticating? • Technical details (e.g. p53 is hard to measure, multiple non-equivalent lesions, etc). • To be of clinical value, a prognostic variable has to be really good (easy to determine, cheap to measure, reproducible, etc etc). • Most interesting scientifically: these lesions are the sine qua non of the cancers themselves.

6. Comparing apples with apples: Proliferation / Aggessive growth RAS RAF • RAS mutations (20%) not prognostic in melanoma (1993). • Almost all (>90%) melanoma has a RAS or RAF mutation (2002). • But did we learn something—yes, B-RAF inhibitors might make an excellent melanoma therapy.

7. If the obvious candidates don’t work, what does? • Things that can be measured: • Easily • Cheaply • Non-invasively • Reliably • Things that help discern very dissimilar entities (e.g. the pathologist’s friend). • Things that we identified empirically.

8. 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).

9. What would you do when faced with sick child, frightened parents, unsure pathologists (not to mention greedy malpractice attornies, 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

10. 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

11. 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

12. 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).

13. 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 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’).

14. 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 poor. • Important Observation: pediatric cancers tend to have simple cytogenetics, while adult cancers are more complex

15. SKY Visible Enhanced Cytogenetics 2003: Chromosome painting and Spectral karyotyping (SKY) Specific Paints (DAPI counterstain)

16. 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.’

17. 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 Ph+ positive). • Treated with hi-dose chemotherapy, total body irradiation, and BMT. • Cytogenetic remission in bone marrow at 6 months post-BMT.

18. BMT 1 month 3 month 6 months 6.5 months ++++ 0 0 +++ 12 months 0 Donor lymphocyte infusions begun PCR on the blood for BCR-ABL • One problem: Low copy Bcr-Abl can be found in ‘normal’ people at modest frequency (carpe diem).

19. Minimal Residual Disease BMT 1 month 3 months 6 months 6.5 months 12 months Bcr-Abl ++++ 0 0 +++ 0 # CA cells 1010 102 104 106 106 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, angiogenesis inhibitors, monoclonal antibodies, etc) in this setting.

20. Amplifications and Deletions: • Done by cytogenetics, comparative cytogenetic hybridization, Southern blot, LOH assay, rarely quantitative PCR. • Adult carcinomas characterized by wholesale gains and losses. • More of scienitific interest than clinical utility • A few examples: N-myc copy # important in neuroblastoma, 14q deletion adverse in colon cancer

21. Tumor A B C D E a b e X Loss of heterozygosity Somatic cells • How tumor suppressor genes have classically been found. • Gives related but different result from CGH (which measures copy number). A B C D E a b c d e X X

22. Comparative Genomic Hybridization • Label tumor DNA with green chrome, label normal DNA red and mix. • Hybridize metaphase spreads with labeled DNA mix. Perform fluorescence microscopy. • Areas of normal copy number are yellow, tumor amps are more GREEN, dels are more RED. • Computer sums results from several metaphases.

23. CGH: Small cell lung cancer From Charite, Humboldt University Berlin

24. 1 2 3 4 5 6 7 8 9 10 11 12 Chr. p q Array CGH • Done in the same manner as conventional CGH, except hybridize >1K feature microarrays of mapped DNA fragments (e.g. BACs) instead of metaphase spreads.

25. 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)

26. IHC / ELISA / Flow • Conjugated antibodies bind cognate antigen (e.g. CD3 on T-cells, neuron specific enolase as neuroendocrine marker) • Ab binding detected by fluorescence or chemical reaction (e.g. horseradish peroxidase)

27. Cyclin E and Breast Cancer • Cyclin E / cdk2 complex is major activity that phosphorylates RB and leads to G1-traversal (i.e. cell divides). • Despite this important role in regulating proliferation, the data that cyclin E are an oncogene or important in cancer are mixed. • Recent NEJM article (Keyomarsi et al. 2002) suggests proper measurement of cyclin E levels is of high prognostic value.

28. Cyclin E and Breast Cancer • If correct, obviously we’d try most aggressive therapy in patients with high cyclin E • Problems: WB not simple to do in a clinically useful way. • ?Publication bias (this single gene is better prognosticator than 10,000 gene microarray).

29. Tissue Microarrays • Little pieces of tumor are embedded in paraffin in microarray format. • Each tumor piece is hybridized to different primary antibody • Can assess hundreds of IHCs simultaneously From www.yalepath.org/DEPT/research/YCCTMA

30. An interesting observation about gene expression tests: • Usually, we measure genes that are pathologically unimportant (CD3, vimentin, etc); to help determine tissue of origin. • We are beginning, however, to have tests for pathogenic molecules (e.g. Her2-neu in breast cancer). • Even better, some of these molecules are good targets for humanized monoclonal antibody therapy (e.g. anti-CD20 = Rituxan).

31. 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 is still used to tell AML from ALL (although now can be done using an antibody to MPO) -Some intrepid member of the audience should now ask me about TELOMERASE.

32. Telomerase activity as tumor marker • Telomerase is an RNA containing enzyme complex that maintains the protective cap (‘telomere’) at the end of chromosomes. • Telomerase activity can be measured by the TRAP assay (requires intact RNA and PCR). • Most cancers express telomerase. • Normal somatic cells don’t express telomerase (so presence of this activity has been proposed as an early diagnostic test for cancer). • Some tumors don’t express telomerase, but still maintain telomere length (‘ALT’).

33. +ALT +telomerase Mouse Lung +ALT Telomerase as marker of pathogenic potential • Tail vein metastasis assay: Inject tumor cell lines intravenously into mice. • Sacrifice animal and count tumors in the lung 6 weeks later. • All lines are ‘malignant’, assay rather measures ability to seed and grow in lung (?metastasis?).

34. ALT+ Survival Telomerase+ Time (years) Telomerase and Glioblastoma • GBM is one of the worst of all adult cancers • Generally, 50% survival measured in weeks. • ALT+ (telomerase negative) tumors may do exceptionally well. • Even if correct, does not necessarily mean telomerase inhibitors will be of therapeutic value.

35. 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.

36. 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.

37. Metastasis-free survival: From van de Vijver et al. NEJM 2002

38. What is the best microarray technology? • RNA expression profiling: most mature in terms of clinical applications. RNA-based assay. Lot of Big Pharma interest. But…Lots of important molecules regulated at protein level (e.g. p53), reproducibility issues. • aCGH: Uses DNA, clinicians are very comfortable using cytogenetic data for therapeutic decisions. “Normal” is always known. Quantifies tumor genomic instability. Sometimes the locus may be more important than the gene (e.g. del 3p). But…less data than expression profiling • Tissue microarrays: Hardest to do well, confounded by tumor heterogeneity (I wouldn’t invest here…). • Proteomics: Most technically difficult at present, but also least mature. Can be done on the serum (instead of the tumor). Immense promise.

39. A Caution: • Medicine >< Science. • Medicine (appropriately) is very conservative and moves much more slowly than science. • 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.

40. 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.

41. 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).

42. 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 skepticism and vastly increased cost and toxicity, thousands of women were treated in this way. • MA required insurance companies BY LAW to pay for ASCT in women with high risk breast cancer. • 2001: Three large randomized trials showed no (or at best very limited) benefit of ASCT. • 2001: Bezwoda article was retracted after auditors concluded the results had been FABRICATED.

43. Summary: Clinical Cancer Genetics • In cancer care, good pathology is crucial and difficult. • Clinical cancer genetics, in 2003, is comprised of cytogenetics, detection of chromosomal translocations and amps/dels, and limited assays of gene expression (IHC, ELISA, flow). • Crucial for diagnosis, therapy, prognostication, and assessment of minimal residual dz. • RNA expression profiling, aCGH, and tissue microarrays hold great promise.

44. A little less conversation, a little more action… If interested in cancer biology, now’s the time to hit the lab (even if all you want to do is go to med school). Ned Sharpless 966-1185 NES@med.unc.edu