Predictive Testing in Non-Small Cell Lung Cancer

2. About US LABS and Genzyme Genetics We offer innovative, high quality testing methods combined with connectivity solutions that help practices operate more efficiently Our pathologists bring a broad range of experiences and outstanding abilities to approach diagnostic challenges We offer a comprehensive menu of pathology and esoteric testing services including: Pathology and Morphology Flow Cytometry Cytogenetics Fluorescence in situ Hybridization Molecular Assays Molecular Genetics Immunohistochemistry

3. Value of Laboratory Testing

4. Prognostic: a parameter that impacts on patients� outcome independent of treatment Predictive: a parameter that is useful to make therapeutic decisions; i.e., outcome depends on the treatment chosen Example: Female gender is prognostic of improved survival in NSCLC. Female gender is predictive of improved response to EGFR-TKIs. Predictive and/or Prognostic Considerations

5. Market Forces � Predictive Testing The majority of drugs under development are for cancer The success of therapeutics such as Herceptin� and Erbitux� have demonstrated the viability of targeted therapies

6. Market Forces � Predictive Testing In a recent survey of pharmaceutical executives: 100% of respondents reported that they utilize biomarkers and other personalized medicine tactics when they evaluate compounds in the discovery phase. Between 12% and 50% of respondents� drug pipelines are personalized therapies. 50% of clinical trials collect DNA from participants to identify biomarkers correlating to drugs� efficacy and safety. 10% of the drugs in late-stage clinical trials have a companion diagnostic.

7. Market Forces � Conclusions The number of available cancer therapeutics will continue to increase The number of companion diagnostics will continue to increase The treatment of cancer will grow more complex Continued expansion of standardized treatment guidelines (eg, NCCN guidelines) Opportunities for laboratories to provide not just results but also decision support

8. Challenges for bringing tests to market Adapting a �clinical trial test� into a �companion diagnostic� Sample type, turnaround time not compatible with current practice Still waiting on FDA Guidance beyond draft publications Confusing reporting metrics (what does equivocal mean?) Most tests were developed in a vacuum - no prospective clinical trials evaluating multiple predictive markers simultaneously

9. What Makes an Effective test? Unmet medical need Tells us something we don�t know Provides clear benefit to the patient Provides a clear answer Tells us what to do next Dovetails with current clinical practice Works with standard samples/biopsies Tells us what we need to know quickly Compatible with existing workflow Time saving or time neutral

10. Non Small Cell Lung Cancer Third most common cancer in the US Leading cause of cancer deaths in men and women More than 75% of cases are diagnosed at an advanced stage Many therapy choices, but most utilize chemotherapies Overall Many possible therapies Most of them are toxic Time is limited Predictive diagnostics can help inform treatment decision

12. Overview of Predictive Analyses used in NSCLC First-line/Maintenance therapy Avastin (bevacizumab) and ALIMTA (pemetrexed) Chemotherapy Platinum-based drugs Gemcitabine ALIMTA Biologic Small molecule, EGFR-directed TKI: Tarceva and Iressa ALK-directed crizotinib

13. What Type of NSCLC?

14. ALIMTA, Histology and Survival

15. ALIMTA, Histology and Survival

16. Avastin and Histology A Phase II clinical trial was designed to investigate the efficacy and safety of bevacizumab plus carboplatin and paclitaxel in patients with advanced or recurrent non�small-cell lung cancer. An analysis of the Safety and Toxicity Results revealed bleeding as the most prominent adverse event and manifested in two distinct clinical patterns minor mucocutaneous hemorrhage major hemoptysis The major hemoptysis events were associated with squamous cell histology Avastin approved in 2006 for non-squamous NSCLC

17. Conclusions - Avastin/ALIMTA AVASTIN (bevacizimab) associated with fatal hemoptysis when directed against squamous cell carcinoma of lung ALIMTA (pemetrexed) generally ineffective against squamous carcinoma. Result Pathology testing and interpretation (H&E and IHC) should be employed to resolve NSCLC into its subtypes If a �significant component� of the lung tumor is squamous cell carcinoma, then AVASTIN or ALIMTA should NOT be used.

18. Challenges Limited Sample About 120,000 cases of unresectable NSCLC each year in the U.S. are diagnosed on small biopsies and cytologies (CAP Today, 2010) Poorly differentiated NSCLC > 20% of samples come back �NSCLC, NOS� IHC stains can be added to increase confidence (Ring BZ et al. Mod Pathol 22:1032, 2009) TTF1+TP63 3.5% misclassified; 22% unclassifiable MUC1, CEACAM5, SLC7A5, CK5/6, TRIM29 4.1% misclassified; 11% unclassifiable

19. ERCC1, RRM1 and TS

20. NSCLC Chemotherapies Platinum Doublet (Cisplatin, Oxaliplatin or Carboplatin) Platinum + Gemcitabine (Gemzar�) Platinum + Vinorelbine (Navelbine�) Platinum + Docetaxel (Taxotere�) Platinum + Paclitaxel (Taxol�) Platinum + Pemetrexed (ALIMTA) Non-Platinum Doublet Gemcitabine (Gemzar) + Paclitaxel (Taxol) Gemcitabine (Gemzar) + Pemetrexed (ALIMTA) Gemcitabine (Gemzar) + Docetaxel (Taxotere)

21. Predictive Tests for First Line/ Maintenance Chemotherapy Platinum Drugs ERCC1 Gemcitabine RRM1 ALIMTA TS (thymidylate synthase)

22. ERCC1 and Platinum-Based Drugs ERCC1=Excision Repair Cross-Complementation Group 1 Mechanism of Platinum-Based Drugs Nucleotide Damage Dilemma of Platinum-Based Drugs Highly Toxic Modest Effect Mechanism of ERCC1 Repairs Nucleotide Damage

23. Pivotal ERCC1 Study Despite undergoing complete resection 33% of patients with pathological stage IA die within 5 years, as do 77% of those with pathological stage IIIA. Adjuvant chemotherapy modestly prolonging 5 yr survival from 4 to 15%, but not without serious adverse effects. International trial (IALT Bio) to evaluate ERCC1 expression and possible benefit from adjuvant cisplatin-based chemotherapy (Stage I through III NSCLC) 761 tumors, ERCC1 expression determined using IHC was positive in 335 (44%) and negative in 426 (56%). PROGNOSTIC BENEFIT 5-year overall survival in patients with no therapy, 46% for ERCC1-positive tumors vs 39% for ERCC1-negative tumors; adjusted HR, 0.66; 95% CI,0.49�0.90; P = 0.009 PREDICTIVE BENEFIT High ERCC1 did not benefit from adjuvant cisplatin-based therapy (HR = 1.14; 95% CI, 0.84�1.55; P = 0.4), whereas patients with low ERCC1 expression received substantial benefit (HR = 0.65; 95% CI, 0.50�0.85; P = 0.002)

24. IALT-Bio: ERCC1 & Adjuvant Cisplatin-Based Chemotherapy ERCC1 Negative = benefited from cisplatin chemotherapy compared to observation (HR = 0.65) (p < 0.002) ERCC1 Positive = no benefit from cisplatin chemotherapy compared to observation (HR = 1.14) (p = 0.40)

25. Clinical Application of ERCC1 Data Stage, age and co-morbidities play an important role in treatment selection In young patients with good performance status, cisplatin may be favored regardless of ERCC1 status ERCC1 will play a major role when the risk/benefit ratio is uncertain i.e. older patients, co-morbidities and stage 1B patients

26. RRM1 and Gemcitabine RRM1 = Ribonucleotide Reductase Subunit 1 Mechanism of Gemcitabine Targets RRM1 for inactivation

27. Clinical Trials � RRM1 Expression RRM1 expression is the dominant cellular determinant of gemcitabine efficacy

28. Clinical Trials � RRM1 and ERCC1 Expression Phase III trial of gemcitabine +/- carboplatin Expression values significantly and inversely correlated with drug response

29. RRM1, ERCC1 and Prognosis

30. Clinical Application

31. TS and ALIMTA Mechanism of ALIMTA ALIMTA is an antifolate that inhibits multiple enzymes involved in purine and pyrimidine synthesis TS is the primary target of ALIMTA TS appears to be expressed at much higher levels in squamous lung tumors than adenocarcinomas TS mRNA levels are predictive of response to ALIMTA/gemcitabine chemotherapy2

32. Challenges Multiple methodologies Clinical trials utilized IHC and/or RT-PCR to quantify mRNA levels Defining a cutoff for �high� vs. �low� expressers Not yet incorporated into professional guidelines NCCN includes RRM1 and ERCC1 as biomarkers of interest, but not yet incorporated into treatment algorithms Limited sample Few labs offer these tests

33. EGFR (Epidermal GrowthFactor Receptor) and KRAS EGFR: Large growth promoting protein lying within the cytoplasmic membrane External domain of the protein binds growth factors and is the target of large monoclonal antibody drugs such as ERBITUX� (cetuximab) and Vectibix� (panitumumab) Internal domain, including the tyrosine kinase domain, is the target of small molecule drugs such as Tarceva (erlotinib) and Iressa (gefitinib) KRAS Functionally lies downstream of EGFR EGFR when activated by growth factors in turn activates KRAS to promote growth

36. EGFR Mutations

37. EGFR Amplification in NSCLC Occurs in 9-23% of all NSCLC patients Does not track with any clinical subset1,2 Associated with increased response rate (RR), TTP and survival in patients treated with TKIs3 Definition and method of copy number/amplification or FISH + varies among studies

38. KRAS Mutations: Mechanisms KRAS gene located on chromosome 12p Mutations are confined to codons 12 (majority) and 13 (remainder) of exon 2 of the KRAS gene Mutations in KRAS are generally a single nucleotide polymorphism (SNP), often a purine/pyrimidine exchange, within one codon Unlike many mutations that inhibit the functioning of a gene, KRAS mutations render the KRAS protein constantly �on� stimulating growth independent of outside effectors, including EGFR As a result, all manipulations of EGFR, through either antibody or small molecule inhibitors, have no ability to inhibit the permanently active mutated KRAS

39. KRAS Mutations in NSCLC KRAS mutations occur frequently in NSCLC 15-30% of NSCLC Mostly in lung adenocarcinoma, rarely in squamous cell carcinoma Associated with smoking history Associated with poor prognosis and rapid disease progression Confer primary resistance to treatment with targeted tyrosine kinase inhibitors TKIs (Tarceva, Iressa) Supersede TKI-sensitizing mutations in EGFR Mutually exclusive with EGFR mutations The presence of KRAS mutations is a poor prognostic indicator in NSCLC regardless of treatment type

40. Prospective Studies of EGFR TKI, EGFR Mutations in Advanced NSCLC

41. Analyses of KRAS Mutations and Efficacy of EGFR TKIs in NSCLC

42. EGFR Gene Copy Number in PredictingClinical Benefit from EGFR TKI Therapy

43. Challenges Turnaround time of up to 2 weeks is often too long to delay treatment decisions Multiple methodologies EGFR sequencing detects more mutations, but is slower PCR detection (DxS) is faster, but detects fewer point mutations Limited samples often lead to QNS results

44. ALK by FISH

45. EML4-ALK: a New Marker Emerges at ASCO 2009 ALK = Anaplastic Lymphoma Kinase Translocations of ALK have been identified in 40% to 60% of anaplastic lymphomas and in B-cell lymphomas, NPM is the most common fusion partner of ALK The fusion of the ALK gene with Echinoderm Microtubule-associated protein-Like 4 (EML4) is associated with lung cancer 3% to 7% of lung tumors have ALK rearrangements ALK rearrangements have also been associated with: light smokers (10 pack years) or never smokers\ a lack of EGFR or KRAS mutations Men lack of response to Tarceva younger age adenocarcinomas with acinar histology

46. ALK Positive NSCLC

47. ALK in the news Crizotinib, an ALK / cMET inhibitor under development by Pfizer, shows exceptional promise in early stage clinical trials Results from a Phase 1/2 clinical trial were presented at ASCO 2010 and generated significant media response 47 of 82 patients had a partial or complete response for an overall response rate of 57%; an additional 33% had stable disease

48. ALK-EML4 Surprises All 82 were positive for ALK rearrangement by FISH, but.. In 29 patients tested for ALK-EML4 by PCR, 31% tested negative for this particular rearrangement Preliminary results also suggest that ALK partial deletions are common Testing for ALK-EML4 alone may miss a significant number of ALK-positive cases

49. Challenges Nomenclature From the NCCN meeting - "only 1 or 2 commercial labs in the whole country that do the test" for EML4-ALK In actuality, many labs do offer it, but its called ALK rearrangement Multiple methodologies FISH is the standard for ongoing clincial trials and will likely be the companion diagnostic RT-PCR detection is more sensitive, but may miss many (as-of-yet undescribed) variants Limited sample

50. NCCN Guidelines � Update for NSCLC Non-Small Cell Lung Cancer NCCN Guidelines recommend that molecular diagnostic studies be included in the pathologic evaluation of NSCLC. Histologic type should be established before EGRF testing EGFR mutation testing should be performed on adenocarcinoma, large cell carcinoma, and NSCLC not otherwise specified Testing is not recommended for squamous cell carcinoma, which has a mutation rate of ~3.6% KRAS mutations are associated with intrinsic TKI resistance, and KRAS testing should be used for the selection of patients as candidates for TKI therapy EGFR mutations are generally exclusive of ALK rearrangements; ALK testing should not be considered for those who test positive for a mutation

51. Interpreting Predictive Test Results (Advanced NSCLC) � Initial Treatment

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