Diagnostic Molecular Pathology Update

1. Diagnostic Molecular Pathology Update Christopher D. Gocke, M.D. Associate Professor of Pathology & Oncology Johns Hopkins Medical Institutions Baltimore, MD

2. Disclosures Christopher D. Gocke, M.D. has no relevant disclosures

3. Recent advances • EGFR testing in lung cancer • New HPV testing recommendations • Expression analysis of breast cancer • Warfarin side effects prediction • HCV genotyping • Molecular screening for MRSA • MPDs defined by JAK2 mutation

4. EGFR testing in lung cancer

5. The EGFR tyrosine kinase pathway ErbB receptor family: ERBB1 (EGFR) ERBB2 (HER2) ERBB3 ERBB4 These form homo- or heterodimers These form only heterodimers Mod Path (2008) 21:S16

6. EGFR in lung cancer • NSCLC (80% of all lung cancers) is a clinical, morphologic and genetic mixed bag • Historically, EGFR is over-expressed in 62% and correlates with poor prognosis • Ligands are also over-expressed  autocrine loop hyperactivity • Small molecule inhibitors and antibodies

7. Hydrophobic pocket Erlotinib EGFR therapy in lung cancer gefitinib and erlotinib reversibly block the kinase region of the ERBB proteins Nature Rev Drug Disc (2004) 3:1001

8. EGFR inhibitor therapy • Early (phase II) trials • Response in previously treated patients:9-19%, median survival 6-8.4 mo (no benefit over chemo alone) • But, some subgroups did better • Women (19% response vs 3% men) • Adenocarcinoma histology • Asian ethnicity • Never smoker (36% vs 8%)

9. Why the better response? • Acquired, somatic mutations in EGFR • Most responding patients had heterozygous mutations • Mutations result in increased receptor activation (phosphorylation) NEJM (2004) 350:2129

10. Mutations are clinically significant • Gain-of-function mutations in 77% of responders vs 7% of refractory patients • 10-20% responders without EGFR mutations indicate other molecular causes (e.g., EGFR amplification or mutations in other ERBB family members

11. EGFR mutations Nat Rev Cancer (2007)7:169

12. Resistance to EGFR inhibitors • Develops after 6-12 months in most • About ½ acquire a T790 exon 20 mutation • Altered EGFR trafficking, active drug excretion are possible alternatives • 2nd generation drugs are targeting irreversible EGFR binding and other ERBB family members

13. Current recommendations for molecular assays • FFPE tissue of adenocarcinoma only (but not mucinous BAC); may need microdissection • Direct sequencing is gold standard, but mutation-specific methods may be OK • Several replicates should be run • Heterogeneity exists in tumor and metastasis—be aware JCO (2008)26:983

14. Direct sequencing of EGFR mutations NEJM (2004)350:2129

15. Other considerations for EGFR testing • EGFR mutant pts treated with placebo do better than controls—a prognostic marker • IHC for protein positive in 50-90% of NSCLC—1 study (BR.21) suggests survival benefit—very inconsistent scoring • Amplification by FISH/RT-PCR positive in 31-45%—significant overlap with mutation positive—unclear how important • KRAS and EGFR mutations mutually exclusive—KRAS positive are inhibitor resistant

16. New HPV recommendations

17. Infectious disease testing: Improving the Pap smear • Relative risk of developing high grade dysplasia (premalignancy) if infected with high risk HPV is increased 76-fold Univ Utah

18. Guidelines for management of abnormal cervical screening tests • Consensus statement guided by American Society for Colposcopy and Cervical Pathology • Am J Obstet Gynecol (2007) 197:346 • Only testing for high-risk (oncogenic) types of HPV is indicated

19. 2. Hybridize with cRNA 1. Lyse cervical cells 5. Detect label 4. Add labeled anti-DNA/RNA antibodies 3. Bind with anti-DNA/RNA antibodies Infectious disease testing: Improving the Pap smear • Hybrid capture HPV detection (Digene)

20. Management of women withASC-US • Reflex testing (of the ASC-US specimen) for HPV (preferred on cost basis) OR repeat cytology OR colpo • Except: adolescents (≤20 yo), no HPV

21. Management of women with ASC-H • Colposcopy • If negative (no CIN2,3 or greater): • HPV testing at 12 mo OR • Cytology at 6 and 12 mo • Repeat colposcopy if these don’t normalize

22. Management of women with LSIL • LSIL is a good indicator of high-risk HPV infection (76.6%) • Prevalence of CIN2+ in this group is 12-16% • Colpo with biopsy • If negative, manage as for ASC (HPV testing at 12 mo OR cytology at 6 and 12 mo) • Except: • Adolescents: no HPV testing • Postmenopausal: monitor with HPV test or cytology, or go direct to colpo

23. Management of women withHSIL • High rate of HPV positivity and CIN2 or greater (84-97% by LEEP) • Colpo with biopsy—HPV testing plays no role in these patients

24. Management of women withatypical glandular cells (AGC) • High rate of CIN2+ and invasive cancer (3-17%) • HPV testing should be done at colpo, but not as a part of triage • If these women are biopsy negative: • And HPV+, repeat HPV/cytology at 6 mo • And HPV-, repeat HPV/cytology at 12 mo • And HPV unknown, repeat HPV/cyto at 6 mo

25. Screening • Most HPV infections clear spontaneously, therefore only women ≥30 yr should undergo HPV testing

26. Screening (2) • Final guidance on 2 controversial areas: • What is the appropriate interval for rescreening a cyto-/HPV- patient? 3 years (compare to annual Paps); <2% develop CIN3+ over next 10 years (JNCI (2005) 97:1072)

27. Screening (3) • What to do with cyto-/HPV+ patients? • A common phenomenon (58% HPV+ women were cyto- in Kaiser study) • Most (60%) of HPV+ women become HPV- spontaneously over 6 months (BJC (2001) 89:1616) • Risk of CIN2+ in them is 2.4-5.1% Follow-up testing in 12 mo, with repeat positives going to colpo

28. Expression analysis of breast cancer

29. Not uniform consensus Current management guidelines for breast cancer NCCN BINV-6

30. Clinically aggressive Gene expression array • Quick way of examining simultaneous expression of thousands of genes • Several uses: • Class finding (unsupervised clustering): • Basal-like (ER/PR/HER2 negative) • Luminal A & B • HER2 over-expressing • Normal-like (These classes correlate with outcome and response to therapy, but not beyond standard clinicopathologic markers)

31. Gene expression array (2) • Identification of molecular targets for specific therapy (e.g., androgen receptor pathway?) • Prognostication (supervised analysis) • Is there a pattern that separates 2 different pre-identified populations, e.g., metastasis-free survival vs not, or cancer recurrence vs not?

32. Wash Harvest RNA, label Hybridize Read intensity Aggregate data Gene expression array (3)

33. Gene expression array (4) • Problems • Tissue requirements: prefer frozen tissue • Small sample size for validation (hundreds) • Population bias • Gene list instability • E.g., 2 different signatures (Mammaprint and Rotterdam) share only 3 genes, yet identify the same groups of low- and high-risk patients

34. 21 gene RT-PCR assay (Oncotype Dx) • Not an array, but a specific gene panel • FDA exempt (CLIA) for node negative, ER +, tamoxifen-treated • Provides a “recurrence score” from 1-100 • Requires FFPE tissue • Performed at company lab in California

35. 21 gene RT-PCR assay RS <18 RS ≥31 Prognostic Predictive JCO (2008) 26:721

36. 70-gene signature (MammaPrint) • FDA approved for Stage I-II, node-negative, ≤5 cm invasive tumor, ≤60 yr • Predicts risk of distant metastasis in next 5-10 yr • First IVDMIA cleared by FDA (2/07) • Requires fresh preserved tumor with a minimum % tumor • Performed at company lab in Amsterdam

37. MammaPrint signature outcome (without adjuvant therapy) NEJM (2002) 347:1999 “Good” = 87% @ 10 yr “Bad” = 44% @ 10 yr

38. Oncologist (2008) 13:477

39. Warfarin MANAGEMENT

40. Maintenance dose variability Molec Interv (2006) 6:223

41. Warfarin metabolism Molec Interv (2006) 6:223

42. Genetic variation in warfarin related enzymes • CYP2C9 (hepatic metabolizer): • *1 (wild-type), rapid metabolizer, ~80% Caucasian population • *2 and *3, slow metabolizers, 12.2% and 7.9% allele frequencies—heterozygotes take twice or three times as long to achieve steady-state when their daily dose is adjusted down

43. Genetic variation in warfarin related enzymes • VKORC1 (Vit K production) (transcriptional variants) • B (wild-type), high-dose phenotype, 60% allele frequency in Caucasian (wide variation reported) • A, low-dose, 40% Caucasian (most Asians)

44. Clinical variability in warfarin dosing Genet Med (2008) 10:89

45. FDA action on warfarin • 2006: adds a “black box” warning for Coumadin about risk of severe or fatal bleeding • 8/2007: adds notice that genetic variation in CYP2C9 and VKOR1 influence phenotype • 9/2007: clears Nanosphere’s Verigene test for identification of some alleles

46. Nanogold DNA genotyping technology Science (2000) 289:1757

47. Problems with genotyping for warfarin dosing • Most studies use INR as endpoint; little data from randomized studies on direct clinical utility in predicting/preventing bleeding or thrombosis (under-anticoagulation) • Little data in non-Caucasians, children • How to present dosing advice to clinicians • Ethical, legal, social implications