7.1 comprehensive adverse events and
references

potential for this research treatment to offer control of the disease is unknown; however, based on information to date, the possibility of an effect on the progression of disease exists. Benefit cannot be promised nor can the chance of benefit be accurately predicted.  
1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin 2009; 59:225-49. 2. Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Ding S, Byrd DR, Cascinelli N, Cochran AJ, Coit DG, Eggermont AM, Johnson T, Kirkwood JM, Leong SP, McMasters KM, Mihm MC, Jr., Morton DL, Ross MI, Sondak VK. Multivariate Analysis of Prognostic Factors Among 2,313 Patients With Stage III Melanoma: Comparison of Nodal Micrometastases Versus Macrometastases. J Clin Oncol. 2010; 28:2452-9. 3. Desmond RA, Soong SJ. Epidemiology of malignant melanoma. Surg Clin North Am 2003; 83:1-29. 4. Tsao H, Atkins MB, Sober AJ. Management of cutaneous melanoma. N Engl J Med 2004; 351:998-1012. 5. FDA. Databases on the FDA website. wwwaccessdatafdagov/scripts/cder/onctools/druglistcfm 2009. 6. Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, Abrams J, Sznol M, Parkinson D, Hawkins M, Paradise C, Kunkel L, Rosenberg SA. High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin Oncol 1999; 17:2105-16. 7. Bellett RE, Mastrangelo MJ, Laucius JF, Bodurtha AJ. Randomized prospective trial of DTIC (NSC-45388) alone versus BCNU (NSC-409962) plus vincristine (NSC-67574) in the treatment of metastatic malignant melanoma. Cancer Treat Rep 1976; 60:595-600. 8. Middleton MR, Grob JJ, Aaronson N, Fierlbeck G, Tilgen W, Seiter S, Gore M, Aamdal S, Cebon J, Coates A, Dreno B, Henz M, Schadendorf D, Kapp A, Weiss J, Fraass U, Statkevich P, Muller M, Thatcher N. Randomized phase III study of temozolomide versus dacarbazine in the treatment of patients with advanced metastatic malignant melanoma. J Clin Oncol 2000; 18:158-66. 9. Weber JS, O'Day S, Urba W, Powderly J, Nichol G, Yellin M, Snively J, Hersh E. Phase I/II study of ipilimumab for patients with metastatic melanoma. J Clin Oncol 2008; 26:5950-6. 10. Attia P, Phan GQ, Maker AV, Robinson MR, Quezado MM, Yang JC, Sherry RM, Topalian SL, Kammula US, Royal RE, Restifo NP, Haworth LR, Levy C, Mavroukakis SA, Nichol G, Yellin MJ, Rosenberg SA. Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4. J Clin Oncol 2005; 23:6043-53. 11. Dudley ME, Wunderlich JR, Robbins PF, Yang JC, Hwu P, Schwartzentruber DJ, Topalian SL, Sherry R, Restifo NP, Hubicki AM, Robinson MR, Raffeld M, Duray P, Seipp CA, Rogers-Freezer L, Morton KE, Mavroukakis SA, White DE, Rosenberg SA. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 2002; 298:850-4. 12. Dudley ME, Rosenberg SA. Adoptive cell transfer therapy. Semin Oncol 2007; 34:52431. 13. Dudley ME, Wunderlich JR, Yang JC, Sherry RM, Topalian SL, Restifo NP, Royal RE, Kammula U, White DE, Mavroukakis SA, Rogers LJ, Gracia GJ, Jones SA, Mangiameli DP, Pelletier MM, Gea-Banacloche J, Robinson MR, Berman DM, Filie AC, Abati A, Rosenberg SA. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol 2005; 23:234657.  
14. Hocker TL, Singh MK, Tsao H. Melanoma genetics and therapeutic approaches in the 21st century: moving from the benchside to the bedside. J Invest Dermatol 2008; 128:2575-95. 15. Sawyers C. Targeted cancer therapy. Nature 2004; 432:294-7. 16. Flaherty K PI, Sosman J, Kim K, Ribas A, McArthur G, Lee RJ, Grippo JF, Nolop K, Chapman P. Phase I study of PLX4032: Proof of concept for V600E BRAF mutation as a therapeutic target in human cancer. J Clin Oncol 2009; 27: 15 suppl. 17. Carvajal RD CP, Wolchok JD, Cane L, Teitcher JB, Lutzky J, Pavlick AC, Bastian BC, Antonescu CR, Schwartz GK. A phase II study of imatinib mesylate (IM) for patients with advanced melanoma harboring somatic alterations of *KIT.* J Clin Oncol 2009; 27: 15 suppl. 18. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR, Futreal PA. Mutations of the BRAF gene in human cancer. Nature 2002; 417:949-54. 19. Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D, Marais R. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004; 116:855-67. 20. Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, Chen C, Zhang X, Vincent P, McHugh M, Cao Y, Shujath J, Gawlak S, Eveleigh D, Rowley B, Liu L, Adnane L, Lynch M, Auclair D, Taylor I, Gedrich R, Voznesensky A, Riedl B, Post LE, Bollag G, Trail PA. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004; 64:7099-109. 21. Haluska F, Pemberton T, Ibrahim N, Kalinsky K. The RTK/RAS/BRAF/PI3K pathways in melanoma: biology, small molecule inhibitors, and potential applications. Semin Oncol 2007; 34:546-54. 22. Eisen T, Ahmad T, Flaherty KT, Gore M, Kaye S, Marais R, Gibbens I, Hackett S, James M, Schuchter LM, Nathanson KL, Xia C, Simantov R, Schwartz B, Poulin-Costello M, O'Dwyer PJ, Ratain MJ. Sorafenib in advanced melanoma: a Phase II randomised discontinuation trial analysis. Br J Cancer 2006; 95:581-6. 23. Agarwala SS, Keilholz, U., Hogg, D., et al. Randomized phase III study of paclitaxel plus carboplatin with or without sorafenib as second-line treatment in patients with advanced melanoma. ASCO Meeting Abstract 2007; 25:Abstract 8510. 24. Lorusso PM, Adjei AA, Varterasian M, Gadgeel S, Reid J, Mitchell DY, Hanson L, DeLuca P, Bruzek L, Piens J, Asbury P, Van Becelaere K, Herrera R, Sebolt-Leopold J, Meyer MB. Phase I and pharmacodynamic study of the oral MEK inhibitor CI-1040 in patients with advanced malignancies. J Clin Oncol 2005; 23:5281-93. 25. LoRusso PM, Krishnamurthi SS, Rinehart JJ, Nabell LM, Malburg L, Chapman PB, DePrimo SE, Bentivegna S, Wilner KD, Tan W, Ricart AD. Phase I pharmacokinetic and pharmacodynamic study of the oral MAPK/ERK kinase inhibitor PD-0325901 in patients with advanced cancers. Clin Cancer Res; 16:1924-37.  
26. Board RE, Ellison G, Orr MC, Kemsley KR, McWalter G, Blockley LY, Dearden SP,
Morris C, Ranson M, Cantarini MV, Dive C, Hughes A. Detection of BRAF mutations in the tumour and serum of patients enrolled in the AZD6244 (ARRY-142886) advanced melanoma phase II study. Br J Cancer 2009; 101:1724-30. 27. Stahl JM, Sharma A, Cheung M, Zimmerman M, Cheng JQ, Bosenberg MW, Kester M, Sandirasegarane L, Robertson GP. Deregulated Akt3 activity promotes development of malignant melanoma. Cancer Res 2004; 64:7002-10. 28. Tolcher AW, Yap, T.A., Fearen, I., Taylor, A., Carpenter, C., Brunetto, A.T., Beeram, M., Papadopoulos, K., Yan, L., de Bono, J. A phase I study of MK-2206, an oral potent allosteric Akt inhibitor (Akti), in patients (pts) with advanced solid tumor (ST). J Clin Oncol 2009; 27:suppl; abstr 3503. 29. Vukelja S, Richards, D., Campos, L.T., Bedell, C., Hagenstad, C., Hyman, W., Letzer, J., Gardner, L., Sportelli, P., Nemunaitis, J. Randomized phase II study of perifosine in combination with capecitabine versus capecitabine alone in patients with second- or third-line metastatic colon cancer. J Clin Oncol 2009; 27:suppl; abstr 4081. 30. Rao RD, Allred, J.B., Windschitl, H.E., Maples, W.J., McWilliams, R.R., Creagan, E.T., Kaur, J.S., Kottchade, L.A., Gornet, M.K., Pockaj, B.A., Markovic, S.N. . N0377: Results of NCCTG phase II trial of the mTOR inhibitor RAD-001 in metastatic melanoma. J Clin Oncol 2007; 15:Suppl; abstr 8530. 31. Curtin JA, Busam K, Pinkel D, Bastian BC. Somatic activation of KIT in distinct subtypes of melanoma. J Clin Oncol 2006; 24:4340-6. 32. Wyman K, Atkins MB, Prieto V, Eton O, McDermott DF, Hubbard F, Byrnes C, Sanders K, Sosman JA. Multicenter Phase II trial of high-dose imatinib mesylate in metastatic melanoma: significant toxicity with no clinical efficacy. Cancer 2006; 106:2005-11. 33. Tsai J, Lee JT, Wang W, Zhang J, Cho H, Mamo S, Bremer R, Gillette S, Kong J, Haass NK, Sproesser K, Li L, Smalley KS, Fong D, Zhu YL, Marimuthu A, Nguyen H, Lam B, Liu J, Cheung I, Rice J, Suzuki Y, Luu C, Settachatgul C, Shellooe R, Cantwell J, Kim SH, Schlessinger J, Zhang KY, West BL, Powell B, Habets G, Zhang C, Ibrahim PN, Hirth P, Artis DR, Herlyn M, Bollag G. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci U S A 2008; 105:3041-6. 34. Rusnak DW, Lackey K, Affleck K, Wood ER, Alligood KJ, Rhodes N, Keith BR, Murray DM, Knight WB, Mullin RJ, Gilmer TM. The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther 2001; 1:85-94. 35. Spector NL, Xia W, Burris H, 3rd, Hurwitz H, Dees EC, Dowlati A, O'Neil B, Overmoyer B, Marcom PK, Blackwell KL, Smith DA, Koch KM, Stead A, Mangum S, Ellis MJ, Liu L, Man AK, Bremer TM, Harris J, Bacus S. Study of the biologic effects of lapatinib, a reversible inhibitor of ErbB1 and ErbB2 tyrosine kinases, on tumor growth and survival pathways in patients with advanced malignancies. J Clin Oncol 2005; 23:2502-12. 36. Lackey KE. Lessons from the drug discovery of lapatinib, a dual ErbB1/2 tyrosine kinase inhibitor. Curr Top Med Chem 2006; 6:435-60. 37. Qiu C, Tarrant MK, Choi SH, Sathyamurthy A, Bose R, Banjade S, Pal A, Bornmann WG, Lemmon MA, Cole PA, Leahy DJ. Mechanism of activation and inhibition of the HER4/ErbB4 kinase. Structure 2008; 16:460-7. 38. Kumar A, Petri ET, Halmos B, Boggon TJ. Structure and clinical relevance of the epidermal growth factor receptor in human cancer. J Clin Oncol 2008; 26:1742-51.  
39. Rudloff U, Samuels Y. A growing family: Adding mutated Erbb4 as a novel cancer target. Cell Cycle; 9. 40. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers WR, Johnson BE, Meyerson M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; 304:1497-500. 41. Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, Singh B, Heelan R, Rusch V, Fulton L, Mardis E, Kupfer D, Wilson R, Kris M, Varmus H. EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A 2004; 101:13306-11. 42. Takano T, Ohe Y, Sakamoto H, Tsuta K, Matsuno Y, Tateishi U, Yamamoto S, Nokihara H, Yamamoto N, Sekine I, Kunitoh H, Shibata T, Sakiyama T, Yoshida T, Tamura T. Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non-small-cell lung cancer. J Clin Oncol 2005; 23:6829-37. 43. Bence AK, Anderson EB, Halepota MA, Doukas MA, DeSimone PA, Davis GA, Smith DA, Koch KM, Stead AG, Mangum S, Bowen CJ, Spector NL, Hsieh S, Adams VR. Phase I
pharmacokinetic studies evaluating single and multiple doses of oral GW572016, a dual EGFRErbB2 inhibitor, in healthy subjects. Invest New Drugs 2005; 23:39-49.  
44. Burris HA, 3rd, Hurwitz HI, Dees EC, Dowlati A, Blackwell KL, O'Neil B, Marcom PK, Ellis MJ, Overmoyer B, Jones SF, Harris JL, Smith DA, Koch KM, Stead A, Mangum S, Spector NL. Phase I safety, pharmacokinetics, and clinical activity study of lapatinib (GW572016), a reversible dual inhibitor of epidermal growth factor receptor tyrosine kinases, in heavily pretreated patients with metastatic carcinomas. J Clin Oncol 2005; 23:5305-13. 45. Burris HA, 3rd, Taylor CW, Jones SF, Koch KM, Versola MJ, Arya N, Fleming RA, Smith DA, Pandite L, Spector N, Wilding G. A phase I and pharmacokinetic study of oral lapatinib administered once or twice daily in patients with solid malignancies. Clin Cancer Res 2009; 15:6702-8. 46. LoRusso PM, Jones SF, Koch KM, Arya N, Fleming RA, Loftiss J, Pandite L, Gadgeel S, Weber BL, Burris HA, 3rd. Phase I and pharmacokinetic study of lapatinib and docetaxel in patients with advanced cancer. J Clin Oncol 2008; 26:3051-6. 47. Chu QS, Schwartz G, de Bono J, Smith DA, Koch KM, Versola MJ, Pandite L, Arya N, Curtright J, Fleming RA, Ho PT, Rowinsky EK. Phase I and pharmacokinetic study of lapatinib in combination with capecitabine in patients with advanced solid malignancies. J Clin Oncol 2007; 25:3753-8. 48. Chu QS, Cianfrocca ME, Goldstein LJ, Gale M, Murray N, Loftiss J, Arya N, Koch KM, Pandite L, Fleming RA, Paul E, Rowinsky EK. A phase I and pharmacokinetic study of lapatinib in combination with letrozole in patients with advanced cancer. Clin Cancer Res 2008; 14:448490. 49. Medina PJ, Goodin S. Lapatinib: a dual inhibitor of human epidermal growth factor receptor tyrosine kinases. Clin Ther 2008; 30:1426-47. 50. GlaxoSmithKline. Lapatinib [package insert]. GlaxoSmithKline, Research Triangle Park, NC 2007. 51. FDA ApfAnN-. Medical reviews [FDA Web site]. http://wwwfdagov/cder/foi/nda/022059s000TOChtm 2008.  
52. Koch KM, Reddy NJ, Cohen RB, Lewis NL, Whitehead B, Mackay K, Stead A, Beelen AP, Lewis LD. Effects of food on the relative bioavailability of lapatinib in cancer patients. J Clin Oncol 2009; 27:1191-6. 53. Gomez HL, Doval DC, Chavez MA, Ang PC, Aziz Z, Nag S, Ng C, Franco SX, Chow LW, Arbushites MC, Casey MA, Berger MS, Stein SH, Sledge GW. Efficacy and safety of lapatinib as first-line therapy for ErbB2-amplified locally advanced or metastatic breast cancer. J Clin Oncol 2008; 26:2999-3005. 54. Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, Jagiello-Gruszfeld A, Crown J, Chan A, Kaufman B, Skarlos D, Campone M, Davidson N, Berger M, Oliva C, Rubin SD, Stein S, Cameron D. Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med 2006; 355:2733-43. 55. Cameron D, Casey M, Press M, Lindquist D, Pienkowski T, Romieu CG, Chan S, Jagiello-Gruszfeld A, Kaufman B, Crown J, Chan A, Campone M, Viens P, Davidson N, Gorbounova V, Raats JI, Skarlos D, Newstat B, Roychowdhury D, Paoletti P, Oliva C, Rubin S, Stein S, Geyer CE. A phase III randomized comparison of lapatinib plus capecitabine versus capecitabine alone in women with advanced breast cancer that has progressed on trastuzumab: updated efficacy and biomarker analyses. Breast Cancer Res Treat 2008; 112:533-43. 56. Burstein HJ, Storniolo AM, Franco S, Forster J, Stein S, Rubin S, Salazar VM, Blackwell KL. A phase II study of lapatinib monotherapy in chemotherapy-refractory HER2-positive and HER2-negative advanced or metastatic breast cancer. Ann Oncol 2008; 19:1068-74. 57. Blackwell KL, Pegram MD, Tan-Chiu E, Schwartzberg LS, Arbushites MC, Maltzman JD, Forster JK, Rubin SD, Stein SH, Burstein HJ. Single-agent lapatinib for HER2overexpressing advanced or metastatic breast cancer that progressed on first- or second-line trastuzumab-containing regimens. Ann Oncol 2009; 20:1026-31. 58. Iwata H, Toi, M., Fujiwara, Y. et al. Phase II clinical study of lapatinib (GW572016) in patients with advanced or metastatic breast cancer. Abstract; Breast Cancer Symposium; December 14-17; San Antonio, Texas 2006; Abstr. 1091. 59. Di Leo A, Gomez HL, Aziz Z, Zvirbule Z, Bines J, Arbushites MC, Guerrera SF, Koehler M, Oliva C, Stein SH, Williams LS, Dering J, Finn RS, Press MF. Phase III, double-blind, randomized study comparing lapatinib plus paclitaxel with placebo plus paclitaxel as first-line treatment for metastatic breast cancer. J Clin Oncol 2008; 26:5544-52. 60. Crown JP, Burris HA, 3rd, Boyle F, Jones S, Koehler M, Newstat BO, Parikh R, Oliva C, Preston A, Byrne J, Chan S. Pooled analysis of diarrhea events in patients with cancer treated with lapatinib. Breast Cancer Res Treat 2008; 112:317-25. 61. Lacouture ME, Laabs SM, Koehler M, Sweetman RW, Preston AJ, Di Leo A, Gomez HL, Salazar VM, Byrne JA, Koch KM, Blackwell KL. Analysis of dermatologic events in patients with cancer treated with lapatinib. Breast Cancer Res Treat 2009; 114:485-93. 62. Perez EA, Koehler M, Byrne J, Preston AJ, Rappold E, Ewer MS. Cardiac safety of lapatinib: pooled analysis of 3689 patients enrolled in clinical trials. Mayo Clin Proc 2008; 83:679-86. 63. Prickett TD, Agrawal NS, Wei X, Yates KE, Lin JC, Wunderlich JR, Cronin JC, Cruz P, Rosenberg SA, Samuels Y. Analysis of the tyrosine kinome in melanoma reveals recurrent mutations in ERBB4. Nat Genet 2009; 41:1127-32. 64. Gross ME, Shazer RL, Agus DB. Targeting the HER-kinase axis in cancer. Semin Oncol 2004; 31:9-20.  
65. Harari PM, Allen GW, Bonner JA. Biology of interactions: antiepidermal growth factor receptor agents. J Clin Oncol 2007; 25:4057-65. 66. Rosell R, Moran T, Queralt C, Porta R, Cardenal F, Camps C, Majem M, Lopez-Vivanco G, Isla D, Provencio M, Insa A, Massuti B, Gonzalez-Larriba JL, Paz-Ares L, Bover I, GarciaCampelo R, Moreno MA, Catot S, Rolfo C, Reguart N, Palmero R, Sanchez JM, Bastus R, Mayo C, Bertran-Alamillo J, Molina MA, Sanchez JJ, Taron M. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med 2009; 361:958-67. 67. Mok TS, Wu YL, Thongprasert S, Yang CH, Chu DT, Saijo N, Sunpaweravong P, Han B, Margono B, Ichinose Y, Nishiwaki Y, Ohe Y, Yang JJ, Chewaskulyong B, Jiang H, Duffield EL, Watkins CL, Armour AA, Fukuoka M. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009; 361:947-57. 68. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, Gianni L, Baselga J, Bell R, Jackisch C, Cameron D, Dowsett M, Barrios CH, Steger G, Huang CS, Andersson M, Inbar M, Lichinitser M, Lang I, Nitz U, Iwata H, Thomssen C, Lohrisch C, Suter TM, Ruschoff J, Suto T, Greatorex V, Ward C, Straehle C, McFadden E, Dolci MS, Gelber RD. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005; 353:1659-72. 69. Moore MJ, Goldstein D, Hamm J, Figer A, Hecht JR, Gallinger S, Au HJ, Murawa P, Walde D, Wolff RA, Campos D, Lim R, Ding K, Clark G, Voskoglou-Nomikos T, Ptasynski M, Parulekar W. Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 2007; 25:1960-6. 70. LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA. Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resist Updat 2008; 11:32-50. 71. Prickett TD, Agrawal, N.S., Wei, X., Yates, K.E., Lin, J.C., Wunderlich, J., Cronin, J.C., Cruz, P., NISC Comparative Sequencing Program, Rosenberg, S.A., Samuels, Y. Analysis of the tyrosine kinome in melanoma reveals recurrent mutations in ERBB4. Nat Genet 2009; in press. 72. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M, Rubinstein L, Shankar L, Dodd L, Kaplan R, Lacombe D, Verweij J. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009; 45:228-47. 73. Schwartz LH, Bogaerts J, Ford R, Shankar L, Therasse P, Gwyther S, Eisenhauer EA. Evaluation of lymph nodes with RECIST 1.1. Eur J Cancer 2009; 45:261-7. 74. Simon R. Optimal two-stage designs for phase II clinical trials. Control Clin Trials 1989; 10:1-10.
appendix a clinical trials of
references

| Authors                                                                                 | Patient Population                                                                                                                              | No.   | Treatment                                                                                                                                                         | Response                                                                                                                                                                                            |
|-----------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------|-------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Regimens                                                                                |                                                                                                                                                 |       |                                                                                                                                                                   |                                                                                                                                                                                                     |
| Burstein et al.[73]   Phase II   International   OL                                     | Cohort A: HER2-positive MBC  refractory to an anthracycline, a  taxane, capacitabine, and trastuzumab                                           | 140   | Lapatinib                                                                                                                                                         | Response RR: 4.3%; clinical                                                                                                                                                                         |
| 1,500 mg QD                                                                             | benefit: 5.7%;  median TTP: 9.1 wk; median PFS: 9.1 wk;  median survival: 29.4 wk                                                               |       |                                                                                                                                                                   |                                                                                                                                                                                                     |
| Cohort B: HER2-negative  MBC refractory to an anthracycline, a taxane, and capecitabine | No objective responses; median TTP: 7.6 wk; median PFS: 7.6 wk; median survival: 18.6 wk                                                        |       |                                                                                                                                                                   |                                                                                                                                                                                                     |
| 89                                                                                      |                                                                                                                                                 |       |                                                                                                                                                                   |                                                                                                                                                                                                     |
| Blackwell et al.[74]   Phase II   MC, OL                                                | HER2-positive MBC refractory                                                                                                                    | 78    | Lapatinib                                                                                                                                                         | PR: 7.7%; clinical benefit: 21.8%                                                                                                                                                                   |
| to trastuzumab                                                                          | 1,500mg QD                                                                                                                                      |       |                                                                                                                                                                   |                                                                                                                                                                                                     |
| Iwata et al.[75]   Phase II   MC, OL                                                    | HER2-positive MBC refractory  to an anthracycline, a taxane, capecitabine, and trastuzumab                                                      | 45    | Lapatinib                                                                                                                                                         | Clinical benefit: 33.3%                                                                                                                                                                             |
| 1,500mg QD                                                                              |                                                                                                                                                 |       |                                                                                                                                                                   |                                                                                                                                                                                                     |
| Gomez et al.[76]   Phase II   MC, R, OL                                                 | First-line treatment of HER2- positive advanced breast cancer  or MBC                                                                           | 138   | Lapatinib  1,500mg QD  (n=69) or  Lapatinib  500mg BID  (n=69)                                                                                                    | RR: 24%; clinical benefit: 31%; PFS at 6  mo: 43%; no difference in clinical activity  between doses                                                                                                |
| Geyer et al.[77] and  Cameron et al.[78]   Phase III   MC, R, OL                        | HER2-positive locally advanced  breast cancer or MBC that had  progressed with regimens  including an anthracycline, a  taxane, and trastuzumab | 399   | Lapatinib  1,250mg QD +  capacitabine  1,000 mg/m2  PO BID on days  1-14 of a 21-day  cycle or  Capacitabine  1,250mg/m2 PO  BID on days 1- 14 of a 21-day  cycle | Median TTP: 6.2 mo (combination) vs 4.3  mo (capacitabine alone) (HR=0.57; 95% CI,  0.43-0.77; p<0.001); improved PFS with  combination vs capacitabine alone  (HR=0.55; 95% CI, 0.4-0.74; p<0.001) |
| DiLeo et al.[79]   Phase III   MC, R, DB, PC                                            | First-line therapy of HER2- negative or unknown advanced  breast cancer or MBC                                                                  | 580   | Lapatinib  1,500mg QD +  paclitaxel  175mg/m2 IV  q3wk or Placebo +  paclitaxel  175mg/m2 IV  q3wk                                                                | RR: 35.1% vs 25.3%, combination vs  paclitaxel monotherapy, respectively  (p=0.008); clinical benefit: 40.5% vs 31.9%  (p=0.025); no difference in TTP or OS                                        |
| a from Medina PJ, Goodin S. Clin Ther 2008                                              |                                                                                                                                                 |       |                                                                                                                                                                   |                                                                                                                                                                                                     |  
Clinical Trials of Lapatinib in Patients with Advanced or Metastatic Breast Cancera
appendix a clinical trials of
appendix b erbb4 primers

| GENE Exon                                            | Primer Name                         | Non M13 tagged Forward Primer   | Primer Name   | Non M13 tagged Reverse Primer   |
|------------------------------------------------------|-------------------------------------|---------------------------------|---------------|---------------------------------|
| ERBB4 1b                                             | ERBB4-X1b-F                         | GGGGATATGCCATTTGGAC             | ERBB4-X1b-R   | CGGAGTGCCAGAAGGAAC              |
| ERBB4 2                                              | ERBB4-X2-F                          | AGAACTGGGATAGGCTTGTGG           | ERBB4-X2-R    | TTCCAGGTATCAGCACACAGG           |
| ERBB4 3b                                             | ERBB4-X3b-F                         | GGCAACTGTTTGTGTCTTTTCA          | ERBB4-X3b-R   | AAGCATATTTGCCATTTTGGA           |
| ERBB4 4b                                             | ERBB4-X4b-F                         | TTCATCAACAAGCAGTTTGACA          | ERBB4-X4b-R   | TCGCCACATAGGGTAGAACA            |
| ERBB4 5                                              | ERBB4-X5-F                          | AAATCCTCATAAAGGAGCAGGAG         | ERBB4-X5-R    | CCAAAGCAAATCAACCACAAG           |
| ERBB4 6                                              | ERBB4-X6-F                          | TGAATTGAGTCAAAGACAGGGTG         | ERBB4-X6-R    | GGAATGACTTTGAGGAGGGC            |
| ERBB4 7b                                             | ERBB4-X7b-F                         | TGAAAGTAATATTTGCTGTGTTGC        | ERBB4-X7b-R   | TTCTTTTGATTTCAAATAATGACCT       |
| ERBB4 8b                                             | ERBB4-X8b-F                         | TGTTTTGAGCTTGTTTGCTGA           | ERBB4-X8b-R   | AAACCTTGTTATATAGGCCCAGTTC       |
| ERBB4 9b                                             | ERBB4-X9b-F                         | TTGGCCAAAAATAAGTTTCTCAA         | ERBB4-X9b-R   | CACTTTGTAAAATAACTTGCACAAAAA     |
| ERBB4 10b                                            | ERBB4-X10b-F                        | AAATTTGGCTACATCTCTTCTTGA        | ERBB4-X10b-R  | AAATTATATTGTTCATAGCGCAACA       |
| ERBB4 11                                             | ERBB4-X11-F                         | CCTTTCTCACTTCCCAACTTTC          | ERBB4-X11-R   | TACCTCACACCATCATCGGAG           |
| ERBB4 12b                                            | ERBB4-X12b-F                        | TTTTCTCACTTCCCCCTCCT            | ERBB4-X12b-R  | TCCAAAGAAGAATGGGAAAAA           |
| ERBB4 13b                                            | ERBB4-X13b-F                        | TCCCCTTGATTTTGGTGTTT            | ERBB4-X13b-R  | ATGAGGTGAAGGCAACCCTA            |
| ERBB4 14                                             | ERBB4-X14-F                         | TGATGCTCCTGGCACATAGAG           | ERBB4-X14-R   | CCCATGGCATCCTGTAAGTAG           |
| ERBB4 15                                             | ERBB4-X15-F                         | TCTTAGAGGAAGATTTGCCACC          | ERBB4-X15-R   | CATTTCAGAGATGGTACCAGGG          |
| ERBB4 16                                             | ERBB4-X16-F                         | GCTTCCCATGTTCTTCCTCC            | ERBB4-X16-R   | AAGTAAGAAAGTTGGCTTGAGAAGG       |
| ERBB4 17                                             | ERBB4-X17-F                         | TGTGGATAATGTCTTGTACAACTGC       | ERBB4-X17-R   | TTCACAAGCTTTGTTTAACGGAC         |
| ERBB4 18b                                            | ERBB4-X18b-F                        | TTCTTCTTTCCGCTTTGCAG            | ERBB4-X18b-R  | TCCATTGGCTATTATTTTCTAAACA       |
| ERBB4 19b                                            | ERBB4-X19b-F                        | TGTAACAGGTGCTAAATAAACATTTG      | ERBB4-X19b-R  | TGATTGCCTGGGTGTCTGTA            |
| ERBB4 20b                                            | ERBB4-X20b-F                        | TTGAGTTGAAATCATGGTATTGC         | ERBB4-X20b-R  | TTCCATAGAAATTGACAGGCACT         |
| ERBB4 21b                                            | ERBB4-X21b-F                        | GGGAAAACCTGGGCATTAAC            | ERBB4-X21b-R  | TCAAGCAAGATTGCTCTCAAAA          |
| ERBB4 22                                             | ERBB4-X22-F                         | AGGCCAGCCCAAAGACTC              | ERBB4-X22-R   | TAACTGCTTTAGGAAATTAGGCTTATC     |
| ERBB4 23b                                            | ERBB4-X23b-F                        | TTGGTGTTTGGATTGACCTG            | ERBB4-X23b-R  | TGATGGTGATAACATTATTTTGCAG       |
| ERBB4 24                                             | ERBB4-X24-F                         | GAGTCGTTTCTTTCACTAGCTTGC        | ERBB4-X24-R   | TGTTTGTGGTCCTTTCCACAG           |
| ERBB4 25b                                            | ERBB4-X25b-F                        | TGTGTCTGATGGGCAATCTT            | ERBB4-X25b-R  | TTATTTTGAAATGTTAGTGCTTATGAA     |
| ERBB4 26b                                            | ERBB4-X26b-F                        | CCATCATTCCATTTTTCTTTCC          | ERBB4-X26b-R  | AAGCAAAGACCGAAAATCCT            |
| ERBB4 27b                                            | ERBB4-X27b-F                        | ACAACGCCTTCTCTCCACAT            | ERBB4-X27b-R  | AATGGCGATCGTTTCTGAAT            |
| ERBB4 28_1b ERBB4-X28_1b-F TTTTCCAGAAACTAGAGGTTAGCTG | ERBB4-X28_1b-R GGTAGTCTGGGTGCTGAAGG |                                 |               |                                 |
| ERBB4 28_2b ERBB4-X28_2b-F AGGCCGAGGATGAGTATGTG      | ERBB4-X28_2b-R GGAAATTGGAGCAGGTGTGT |                                 |               |                                 |  
| ECOG Performance Status Scale                                                                                                                                                              | Karnofsky Performance Scale                                   |                                                                           |                                                                        |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------|---------------------------------------------------------------------------|------------------------------------------------------------------------|
| Grade                                                                                                                                                                                      | Descriptions                                                  | Percent                                                                   | Description                                                            |
| 100                                                                                                                                                                                        | Normal, no complaints, no evidence  of disease.               |                                                                           |                                                                        |
| Normal activity. Fully active, able                                                                                                                                                        |                                                               |                                                                           |                                                                        |
| 0                                                                                                                                                                                          | to carry on all pre-disease  performance without restriction. | 90                                                                        | Able to carry on normal activity;  minor signs or symptoms of disease. |
| Symptoms, but ambulatory.  Restricted in physically strenuous  activity, but ambulatory and able  to carry out work of a light or  sedentary nature (e.g., light  housework, office work). | 80                                                            | Normal activity with effort; some  signs or symptoms of disease.          |                                                                        |
| 1                                                                                                                                                                                          | 70                                                            | Cares for self, unable to carry on  normal activity or to do active work. |                                                                        |
| In bed <50% of the time.  Ambulatory and capable of all  self-care, but unable to carry out  any work activities. Up and about  more than 50% of waking hours.                             | Requires occasional assistance, but                           |                                                                           |                                                                        |
| 60                                                                                                                                                                                         | is able to care for most of his/her  needs.                   |                                                                           |                                                                        |
| 2                                                                                                                                                                                          | 50                                                            | Requires considerable assistance and  frequent medical care.              |                                                                        |
| In bed >50% of the time. Capable  of only limited self-care, confined  to bed or chair more than 50% of  waking hours.                                                                     | 40                                                            | Disabled, requires special care and  assistance.                          |                                                                        |
| 3                                                                                                                                                                                          | 30                                                            | Severely disabled, hospitalization  indicated. Death not imminent.        |                                                                        |
| 100% bedridden. Completely  disabled. Cannot carry on any  self-care. Totally confined to bed  or chair.                                                                                   | 20                                                            | Very sick, hospitalization indicated.  Death not imminent.                |                                                                        |
| 4                                                                                                                                                                                          | 10                                                            | Moribund, fatal processes  progressing rapidly.                           |                                                                        |
| 5                                                                                                                                                                                          | Dead.                                                         | 0                                                                         | Dead.                                                                  |
appendix c performance status criteria
list of drugs that may

When drugs classified as 'substrates' are co-administered with lapatinib there is the potential for higher concentrations of the 'substrate'. When lapatinib is co-administered with compounds classified as 'inhibitors', increased plasma concentrations of lapatinib is the potential outcome. The co-administration of 'inducers' would potentially lower plasma lapatinib concentrations. Only major substrates and effective inducers are listed.  
| CYP3A4 Substrates  Albuterol  Alfentanil Alprazolam Amiodarone Amlodipine Amprenavir Aprepitant Aripiprazole Atazanavir Atorvastatin Benzphetamine Bisoprolol Bortezomib Bosentan Bromazepam Bromocriptine Budesonide Buprenorphine Buspirone  Busulfan Carbamazepine Cerivastatin Chlordiazepoxide Chloroquine Chlorpheniramine Cilostazol Cisapride Citalopram Clarithromycin Clobazam Clonazepam Clorazepate Cocaine Colchicine Conivaptan Cyclophosphamide Cyclosporine Dantrolene Dapsone Dasatinib (1)  Delavirdine Diazepam  Dihydroergotamine  Diltiazem  Disopyramide  Docetaxel Doxepin Doxorubicin Doxycycline Efavirenz Eletriptan Enalapril Eplerenone Ergoloid mesylates Ergonovine Ergotamine Erythromycin Escitalopram Estradiol Estrogens, conj., synthetic  Estrogens, conj., equine Estrogens, conj., esterified  Estrone Estropipate Ethinyl estradiol Ethosuximide Etoposide Exemestane Felbamate Felodipine Fentanyl Flurazepam Flutamide Fluticasone Fosamprenavir Gefitinib Haloperidol Ifosfamide Imatinib Indinavir Irinotecan Isosorbide Isosorbide dinitrate Isosorbide mononitrate   | Isradipine  Itraconazole Ketamine  Ketoconazole Lansoprazole Letrozole Levonorgestrel Lidocaine Losartan Lovastatin Medroxyprogesterone  Mefloquine Mestranol Methadone Methylergonovine Methysergide  Miconazole Midazolam Miglustat Mirtazapine Modafinil Montelukast Moricizine Nateglinide Nefazodone Nelfinavir Nevirapine Nicardipine Nifedipine Nimodipine Nisoldipine Norethindrone Norgestrel Ondansetron Paclitaxel Pergolide Phencyclidine Pimozide Pipotiazine  Primaquine Progesterone Quetiapine   | Quinidine  Rabeprazole  Ranolazine Repaglinide Rifabutin Ritonavir Salmeterol Saquinavir Sibutramine Sildenafil Simvastatin Sirolimus Spiramycin Sufentanil Sunitinib Tacrolimus Tamoxifen Tamsulosin Telithromycin Teniposide Tetracycline Theophylline Tiagabine Ticlopidine Tipranavir Tolterodine Toremifene Trazodone Triazolam Trimethoprim Trimipramine Troleandomycin Vardenafil Venlafaxine Verapamil Vinblastine Vincristine Vinorelbine Zolpidem Zonisamide Zopiclone   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| CYP3A4 Inhibitors  Acetominophen  Acetazolamide Amiodarone Amlodipine Amprenavir Anastrozole Aprepitant Atazanavir Atorvastatin Azelastine Azithromycin Betamethasone Bortezomib Bromocriptine Caffeine Cerivastatin Chloramphenicol Chlorzoxazone Cimetidine Ciprofloxacin  Cisapride Clarithromycin Clemastine Clofazimine Clotrimazole Clozapine Cocaine Conivaptan Cyclophosphamide Cyclosporine Danazol Dasatinib (1) Delavirdine Desipramine Dexmedetomidine Diazepam   | Diclofenac  Dihydroergotamine  Diltiazem Disulfiram Docetaxel  Doxorubicin Doxycycline Drospirenone Efavirenz Enoxacin Entacapone Ergotamine Erythromycin Ethinyl estradiol Etoposide Felodipine Fentanyl Fluconazole Fluoxetine Fluvastatin Fluvoxamine Fosamprenavir Glyburide Grapefruit juice (2) Haloperidol Hydralazine  Ifosfamide Imatinib Indinavir Irbesartan Isoniazid Isradipine Itraconazole Ketoconazole Lansoprazole Lidocaine   | Lomustine  Losartan  Lovastatin Mefloquine Mestranol Methadone Methimazole Methoxsalen Methylprednisolone Metronidazole Miconazole Midazolam Mifepristone Mirtazapine Mitoxantrone Modafinil Nefazodone Nelfinavir Nevirapine Nicardipine Nifedipine Nisoldipine Nizatidine Norfloxacin Olanzapine Omeprazole Orphenadrine Oxybutynin Paroxetine Pentamidine Pergolide Phencyclidine Pilocarpine Pimozide Pravastatin  Prednisolone   | Primaquine  Progesterone Propofol  Propoxyphene Quinidine Quinine Quinupristin Rabeprazole Ranolazine Risperidone Ritonavir Saquinavir Selegiline Sertraline Sildenafil Sirolimus Sulconazole Tacrolimus Tamoxifen Telithromycin Teniposide Testosterone Tetracycline Ticlopidine Tranylcypromine Trazodone Troleandomycin Valproic acid Venlafaxine Verapamil Vinblastine Vincristine Vinorelbine Voriconazole Zafirlukast Ziprasidone   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| CYP3A4 Inducers  Aminoglutethimide  Carbamazepine Fosphenytoin Nafcillin                                                                                                                                                                                                                                                                                                                                                                                                      | Nevirapine  Oxcarbazepine Pentobarbital Phenobarbital                                                                                                                                                                                                                                                                                                                                                                                           | Phenytoin  Primidone Rifabutin Rifampin                                                                                                                                                                                                                                                                                                                                                                                               | Rifapentine  St. John's wort (3)                                                                                                                                                                                                                                                                                                                                                                                                          |  
Note: Adapted from Cytochrome P450 Enzymes: Substrates, Inhibitors, and Inducers. In: Lacy CF, Armstrong LL, Goldman MP, Lance LL eds. Drug Information Handbook 15TH ed. Hudson, OH; LexiComp Inc. 2007: 1899-1912. Only major substrates and effective inducers are listed. Additional information for drug interactions with cytochrome P450 isoenzymes can be found at http://medicine.iupui.edu/flockhart/.  
(1) Investigator's Brochure: Dasatinib (BMS 354825). Bristol-Myers Squibb. October 2006. (2) Malhotra *et al*. (2001). Clin Pharmacol Ther. 69:14-23. (3) Mathijssen *et al*. (2002). J Natl Cancer Inst. 94:1247-1249. Frye *et al*. (2004). Clin Pharmacol Ther. 76:323-329.
appendix c performance status criteria
appendix f patient medication diary

Appendix 7 Patient Diary  
| Name:                                                                               |            |                |              |           |           |           |           |
|-------------------------------------------------------------------------------------|------------|----------------|--------------|-----------|-----------|-----------|-----------|
| take lapatinib - one pill twice daily - 1 hour before or after breakfast and dinner |            |                |              |           |           |           |           |
| Mon                                                                                 | Tue        | Wed            | Thu          | Fri       | Sat       | Sun       |           |
| 12                                                                                  | 13         | 14             | 15           | 16        | 17        | 18        |           |
| morning [                                                                           | morning [  | morning [      | morning [    |           |           |           |           |
| morning [                                                                           | morning    | morning [      |              |           |           |           |           |
| evening [                                                                           | evening [  | evening [      | evening [    | ening     | evening [ | evening [ |           |
| 23 morning [                                                                        |            |                |              |           |           |           |           |
| 19                                                                                  | morning [  | morning        | morning      | morning [ | evening [ | morning [ | morning [ |
| evening [                                                                           | evening [  | evening [      | evening [    | Lab Work  | evening [ | evening [ |           |
| RM الدع التس                                                                        |            |                |              |           |           |           |           |
| 26                                                                                  | 27         | 28             | 29           | 30        | 1         | 2         |           |
| morning                                                                             | morning    | morning        | morning      | morning | | morning [ | morning | |           |
| evening [                                                                           | evening [  | evening [      | evening      | ening     | evening [ | evening [ |           |
| morning                                                                             |            |                |              |           |           |           |           |
| morning                                                                             | morning    | moming         | moming       | evening [ | morning [ | morning [ |           |
| evening [                                                                           | evening [  | evening [      | evening [    | Lab Work  | evening [ | evening [ |           |
| Clinic visit                                                                        |            |                |              |           |           |           |           |
| List all symptoms whether or not Dates                                              | Treated at | Treated in     | Hospitalized | Comments  |           |           |           |
| you think they are related to the                                                   | Home       | doctors office |              |           |           |           |           |
| lapatinib                                                                           | or Chnic   |                |              |           |           |           |           |
| Diarrhea                                                                            |            |                |              |           |           |           |           |
| Nausea                                                                              |            |                |              |           |           |           |           |
| Vomiting                                                                            |            |                |              |           |           |           |           |
| Other  GI symptoms (describe)                                                       |            |                |              |           |           |           |           |
| Skin rash (describe)                                                                |            |                |              |           |           |           |           |
| Other symptoms                                                                      |            |                |              |           |           |           |           |
appendix g laboratory methodology for
laboratory methodology

Pharmacokinetic Studies Pharmacokinetics of lapatinib Venous blood samples should be collected in a 6-ml sodium heparin (green top) tube at the following time points:
Pre-treatment (before start of lapatinib)
1h, 2h, 3h, 4h, 6h, 8h post 1st dose in the morning (up to pre evening dose) Day 28 (± 5 days) - before 1st dose in the morning and 1h, 2h, 3h, 4h, 6h, 8h post morning dose (up to pre-evening dose).  
For samples collected at the Surgery Branch, NCI, immediately place specimens on wet ice and refrigerate. The date and **exact** time of each blood draw should be recorded on the sample tube and the PK sheet. Please page 102-11964 (Gareth Peters or alternate tech) for immediate pick-up. (Contact the Clinical Pharmacology Program (CPP) processing group in 10/5A09 at 301-5946131 or 301-402-3622 with any questions).
appendix g laboratory methodology for
instructions for specimen processing and

1. Draw specimen in 6ml Na Heparin tube (green top) 2. Place blood tubes on wet ice immediately after draw - keep cold (4C) until aliquots are placed in the freezer. 3. Process samples within one hour of draw whenever possible. 4. Spin at 2400rpm (1250g) for 5min at 4C 5. After centrifugation, split each Na heparin tube sample into 2 aliquots and store aliquots in cryovials (ex. Nunc 375418). 6. Store at -80 C 7. Provide the date and time of draw both on the frozen tube and PK sheet. Provide patient's assigned study code and protocol number on PK sheet. Put patient study code and protocol number on tubes to link them to PK sheet. 8. Do not allow samples to thaw once frozen.
appendix g laboratory methodology for
specimen shipping

1. Place samples in a cardboard sample box with an absorbent strip (ex. Saf-T-Pak \#STP-152 250ml absorbent) 2. Place sample box and PK sheet in a ziplock bag (ex. Lab Guard \# SBL2X810B) 3. Package specimen bag with dry ice inside an insulated shipper (ex. Saf-T-Pak \#STP-300 Category A shipping overpak). Note: Any shipping packaging approved for a Category A substance is acceptable; dry ice is considered a hazardous material and thus must be handled by appropriately trained individuals. 4. Ship via FedEx Priority Overnight to: William Figg National Cancer Institute 9000 Rockville Pike Bldg 10 Rm 5A01 Bethesda, MD 20892 Tel: 301-402-3622 5. Ship only on Mon-Wed. Do not ship the day before a federal holiday. 6. Send PK sheets with shipment. 7. If shipping samples for more than one pt, separate out patient specimens in separate ziplock bags. 8. Notify me via email (comptok@mail.nih.gov) or fax (301 402-8606) on the date of shipment. Provide contact information for the person in charge of shipping at that institute as well as the number of samples shipped.
appendix g laboratory methodology for
sample data collection

All samples sent to the Clinical Pharmacology Program (CPP) will be barcoded, with data entered and stored in the Patient Sample Data Management System (PSDMS) utilized by the CPP. This is a secure program, with access to the PSDM System limited to defined CPP personnel, who are issued individual user accounts. Installation of PSDMS is limited to computers specified by Dr. Figg. These computers all have a password restricted login screen. All CPP personnel with access to patient information annually complete the NIH online Protection of Human Subjects course. PSDMS creates a unique barcode ID for every sample and sample box, which cannot be traced back to patients without PSDMS access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location. Patient demographics associated with the clinical center patient number are provided in the system. For each sample, there are notes associated with the processing method (delay in sample processing, storage conditions on the ward, etc.).
appendix g laboratory methodology for
sample storage and destruction

Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80 C according to stability requirements. These freezers are located onsite in the CPP and offsite at NCI Frederick Central Repository Services (Fisher Bioservices) in Frederick, MD. Visitors to the laboratory are required to be accompanied by laboratory staff at all times. Access to stored clinical samples is restricted. Samples will be stored until requested by a researcher named on the protocol. All requests are monitored and tracked in the PSDM System. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per the IRB approved protocol) and that any unused samples must be returned to the CPP. It is the responsibility of the NCI Principal Investigator to ensure that the samples requested are being used in a manner consistent with IRB approval. Following completion of this study, samples will remain in storage as detailed above. Access to these samples will only be granted following IRB approval of an additional protocol, granting If, at any time, a patient withdraws from the study and does not wish for their existing samples to be utilized, the individual must provide a written request. Following receipt of this request, the samples will be destroyed (or returned to the patient, if so requested), and reported as such to the IRB. Any samples lost (in transit or by a researcher) or destroyed due to unknown sample integrity (i.e. broken freezer allows for extensive sample thawing, etc.) will be reported as such to the IRB. Sample barcodes are linked to patient demographics and limited clinical information. This information will only be provided to investigators listed on this protocol, via registered use of the PSDMS. It is critical that the sample remains linked to patient information such as race, age, dates of diagnosis and death, and histological information about the tumor, in order to correlate genotype with these variables.  
| Table S6. Primers used for PCR amplification and sequencing of RAS and Raf isoforms.   |                                        |                                          |                   |
|----------------------------------------------------------------------------------------|----------------------------------------|------------------------------------------|-------------------|
| sione and                                                                              | Formand Princi                         | Powerse Prime                            | SEQUENCING Prime  |
| OTCATCOATCTCTTCC                                                                       | GTAAAACGACGGGCAGTCAGCTGATGTTCCCATCCTTC | 17777887888889                           |                   |
| AR                                                                                     | GTAAAACGACGGCCAGT                      |                                          |                   |
| AR                                                                                     | 0TAAAACGACGGCCAGTTATCCCTCTGACCTGTTTCC  | AG                                       | 0TAAACGACGGCCAG   |
| ARAF-4                                                                                 | GTAAAACGACGGCCAGT                      |                                          |                   |
| ARAF-S                                                                                 | ASTACCAACCSCCAACACT                    | OTAAAACGACGGCCAGTGAGAAATGAGGTGACTTGCCC   | CTAAAACGACGGCCAG1 |
| ARAF-8                                                                                 | CAAACGACGGGGGTCAACGTCCATATGGTCAGGGG    | OTAAAACGACGGGG                           |                   |
| ARAF-T                                                                                 | GTAAACGACGGCCAGTCCAGATGGCATCTAAC       | 00                                       |                   |
| ARAF-8                                                                                 | CACACTTTCAGCACTGATGR                   | GTAAAACGACGGCCAGT                        |                   |
| GTAAAACGACGGCCAGTAGAAAGTGGTATGCTCGAGG
GTAAAACGACGGCCAGTAGAACAGTGCCACTCCTGATG                                                                                        | TCTCGGTGGATGTTCTTG                     |                                          |                   |
| ARAF-9                                                                                 | GTAAACGACGGCCAG                        |                                          |                   |
| ARAF-11                                                                                | GTAAAACGACGGCCAG                       |                                          |                   |
| ARAF-11                                                                                | OATTTOCCATCATCACACAC                   | GTAAAACG                                 | GTAAAACGACGGCCAG  |
| OTAAACGACGGCCAGTCAAGGTTGTGGATAGTTTCCG
GTAAACGACGGCCAGTAGCAGAGAATCCTCCCAAGTC                                                                                        |                                        |                                          |                   |
| ARAF-11                                                                                | OTAAACGACGGCCAG                        |                                          |                   |
| OTARAACOACOOCCAO                                                                       |                                        |                                          |                   |
| GTAAACGACGGCCAG                                                                        |                                        |                                          |                   |
| GTAAACCACTACTACCAACCAACCACCA                                                           | 00                                     |                                          |                   |
| GTAAACGACGGCCAGTCCCACTOTTACTAGCCAGTCCCAGTTACTAGCCC
GTAAACGACGGCCAGTCTCACTTTATTGAC
GTAAACGACGGCCAGTTTTGCAGTTTGCAGTTTGATTG                                                                                        | CTACAGTATTTCTT                         |                                          |                   |
| BRAF-5
BRAF-8
BRAF-8                                                                                        | OTATION IN TTACTCATCCATATTTCACATTCC
GTAAACGACGGCCAGTTCTGAGTGGTATGATAAGTTATTTCC                                        | GTAAAACGACGGCCA01                        |                   |
| CTAAACGACGG                                                                            |                                        |                                          |                   |
| GTTACTTGAAAGAGGCAAGTATAAAGG                                                            |                                        |                                          |                   |
| ICATCAGAGAACCAGG                                                                       | 0TAAACGACGOCCAOT                       |                                          |                   |
| BRAF:8                                                                                 | OTAAACGACGOCCAGT                       |                                          |                   |
| BRAF-9                                                                                 | GTAAACGACGGCCGTAGAATATGCTT             | GTAAAACGACGGCCAGT                        |                   |
| BRAF-11
ERAF-11                                                                                        | GTAAACGGCCAGTCCATCGAACAACAAGGTTGG      | GTAAAACGACGGCCAGT                        |                   |
| CTAAAACGACGGCCAGT                                                                      |                                        |                                          |                   |
| GTAAACGACGGCCAGTTTAGCATCCTTATGTTCCTGGAC                                                |                                        |                                          |                   |
| BAF-12                                                                                 | OTAAACGACGGCAGTCTCTTCTAATGTATC
GTAAACGACGGCCAGTCATGGATAAATAGGCTTGACTGG                                        | GTAAAACGACGGCCAGT                        |                   |
| ERAF-13                                                                                | CCTATACATGCATGCACAATCR                 | GTAAAACGACGGCCAGT                        |                   |
| ERAF-14                                                                                | TCATCCTAACACATTTCAACC                  | OTAAACOACOGCCAOTTTOTQAATACTOGQAACTATQAAA | OTAAAACGACGOCCAGT |
| ERAF-15                                                                                | GAATTCATCTCGCAATGATGGT                 | AAACGACGGCCAGTTTCACGCTTACG               | 00                |
| BRAF-17                                                                                | CATCTATGTGGCATTGG                      | AAAACGACGGCCAGTCTCAACCC                  | GTAAAACGACGGCCAGT |
| GTAAAACGACGGCCAGTCCACACAAGTGTTCTTTCCTT                                                 | DTAAAACGACGGCCAGT                      |                                          |                   |
| RAF11                                                                                  | GTAAAACGGCCAGTCTGGTCCCATTTTCCTCATC     | ATAGGGGTGTGGAAGGAA                       | GTAAAACGACGGCCAGT |
| RAF1                                                                                   | AGGTATTGGTCATCAGGG                     | GTAAAACGACGGCCAGT                        |                   |
| RAF1                                                                                   | OTARAACGACGGCCAGTGGCCTTGAGCAAATTACCTTC | TOCCTTACTOTAAACAACAGC                    | CTAAACGACGGCCAG   |
| rol 1
ROF 1
ROF                                                                                        | OTAAACGACGGCCADTGGAGGCCAAGAATGAACTTQ   | OTAAAACGACGGCCAG                         |                   |
| TAAAACGACGGCCAGTAAAGCAAGGCATGCTGATTC                                                   | TCCTTGATCAGATTTGAAA                    | GTAAACGAGGGGG                            |                   |
| OTAAACGACGGCCAOTGGTTGTACAGGTAGAGTTTGCC                                                 | OTAAAACOACOOCCAOT                      |                                          |                   |
|                                                                                        | GTAAACGACGGCCAGTTGAAAATCAGCCTTGGAGA    |                                          |                   |
| TTOGCAGGAGGTACTGTTGTTCC                                                                | DTAAAACGACGGCCAGT                      |                                          |                   |
| GATGCAATTCGAAGTCACAC                                                                   | GTAAAACGACGGCCAG                       |                                          |                   |
| OTAAACGACGGCCAGTTGGGTTGATCC
OTAAACGACGGCCAGTTGACAGCAGAACCCAGTGTG                                                                                        | GTAAACGACGGCCAG                        |                                          |                   |
| CTAAACGACGGCCAG                                                                        |                                        |                                          |                   |
| OTAAAACGACGGCCAGTGACAGAACCCACTOTC
GTAAACGACGGCCAGTCCCTGTGTGTAACACTCCTTGGT                                                                                        | OTAAACGACGGGG                          |                                          |                   |
| OTAAAACOACOOCCAO                                                                       |                                        |                                          |                   |
| 00                                                                                     |                                        |                                          |                   |
| OTAAACGACGGCAGTGCAATGCTAACATGGCAAC                                                     |                                        |                                          |                   |
| KRAS-4
NRAS-1
NRAS-1                                                                                        | 0TAAAACGACGACTTCCAATTCCACTCACTACG      | 0TAAACGACGGCCAGT
GTAAACGACGGCCAGT                                          |                   |
| NRAS-1                                                                                 | CAAGAGAGAG                             | CTOCAOTO                                 | 00                |
| TO IN THE DIAGRAM OF CONTROTIC TO TO TO A TAGO
The permer part ad rot mell our quality colories that allow of basis in the battegen have a fired quality score of all was 20 i                                                                                        | CTAAACGACGGCAG                         |                                          |                   |
appendix g laboratory methodology for
appendix h primers used for

Clinical Molecular Profiling Core Genetics Branch, CCR, NCI
50 South Drive, MSC 8000 Building 50, Room 5140 Bethesda MD 20892-8000 CLIA \# 21D1092530
appendix g laboratory methodology for
dna pcr and sequencing assays

Overview: This standard operating procedure describes the technical details of Sanger sequencing of an extant DNA sample produced using the laboratory's CLIA certified protocol. The procedure for DNA extraction from anatomic pathology specimens for clinical testing is described elsewhere. Details regarding the Sanger assay sensitivity, specificity, and validation for particular mutations being tested for are also provided elsewhere.
appendix g laboratory methodology for
workflow

1) DNA analytes are pipetted into a 96-well plate in a pre-PCR environment where the DNA
is safe from contamination.  
2) Perform polymerase chain reaction (PCR) using the selected CLIA-certified test primers. 3) Purify amplified DNA in preparation for sequencing reaction. 4) Run the sequencing reactions in order to produce uniform peak heights, optimize the signal balance, and produce long reads.  
5) Perform second purification to remove any unincorporated dye terminators. 6) Set up the Applied Biosystems 3730 DNA Analyzer, and set the program for the selected plates.  
7) Run the 3730 DNA Analyzer and analyze the DNA sequence data using the software program Variant Reporter™.
appendix g laboratory methodology for
reagents & supplies

A. Components of Applied Biosystems Mix: 1. 2X AmpliTaq Gold Master Mix (Applied Biosystems Cat. \# 4327059) 2. Ultra Pure Glycerol (Invitrogen Cat. \# 15514-011) B. PCR:
1. 5-10 ng/ l DNA 2. Selected 0.6 m Primers (diluted from 100 μM Primers) 3. Water, ultra pure, unfiltered, deionized by reverse osmosis, endotoxin tested, one liter (Quality Biological, Inc. Cat \#: 118-162-131)
4. 2X AmpliTaq Gold Master Mix (Applied Biosystems Cat. \# 4327059)
5. Ultra Pure Glycerol (Invitrogen Cat. \# 15514-011)
C. PCR Purification:
1. Exo-Sap It (USB Cat. \# 78205)
D. Sequencing Reaction:
1. Ultra pure deionized H2O (Quality Biological, Inc., Cat. \# 118-162-131)
2. BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Cat.  
\# 4337456)
3. Hi-DiTM Formamide (Applied Biosystems Cat. \# 4311320)
4. BigDye® Terminator v1.1, v3.1 5X Sequencing Buffer (Applied Biosystems Cat. \# 4336699)
E. Purification:
1. BigDye XTerminator Purification Kit (Applied Biosystems, Cat. \#
4376484)
F. General Supplies:
1. MicroAmp® 96-well Reaction Plate (Applied Biosystems Cat. \# N8010531)
2. MicroAmp® Optical Adhesive Film (Applied Biosystems Cat. \# 4311971)
appendix g laboratory methodology for
equipment

A. ABI 3730 (Applied Biosystems Cat. \# 4373905/4364619/4364620) B. SORVALL Legend RT Plus Centrifuge (Thermo Scientific Cat. \# 75004373) C. Thermo Savant DNA 120 SpeedVac® D. Alpha Innotech FluorChem® 8900 E. PTC-225 DNA Engine Tetrad Thermal Cycler (BioRad {formerly MJ Research} Cat. \#
PTC-0225)
F. Illumina High-Speed Microplate Shaker (VWR International, catalog \# 13500-890).
appendix g laboratory methodology for
assay procedure

For DNA extraction and primer design, please see the relevant SOPs.
appendix g laboratory methodology for
i. pcr reaction protocol

1) Add three uL of PCR Primer set (0.6 uM) into each well. Dry down primer plates. 2) Add the PCR Master Mix. Mix with corresponding DNA to primer plates 3) Follow recipes in Excel Spreadsheet (see file on CMPC shared folder named
"3730_Meltzer_Template_96well(current).xls")  
(uL) 160 10 2 X 1 X Amplitaq Gold PCR Master Mix, Lot # ____________________  
(uL) 0 0 0.6 uM 0.24 uM RSA primers, diluted 50%, robot disp 3ul -dried down, (uL) 16 1 10 ng/ul 10 ng Template DNA (5ng/ul) - robot dispensed 2ul  
(uL) 51.2 3.2 50 % 8 % Glycerol (Invitgoren only), Made ___/___/___ by _____________  
(uL) 92.8 5.8 H2O (uL) 320 20  
| n=        | 16   | n=1   | Stock   | Unit   | Final   |
|-----------|------|-------|---------|--------|---------|
| (uL) 92.8 | 5.8  | H2O   |         |        |         |  
4) Select "VSEQR" on the DNA Engine Tetrad / PTC-225 Peltier Thermal Cycler.  
5) Run a 1-2% Agarose Gel (See 1-2% Agarose Gel Electrophoresis protocol)
appendix g laboratory methodology for
ii. pcr purification protocol

| PCR Cycling         | vseqr 96   | 5 min   |
|---------------------|------------|---------|
| 40 cycles           | 94         | 30 sec  |
| 60                  | 45 sec     |         |
| Thermal cycler used | 72         | 45 sec  |
| ___________________ | 72         | 10 min  |
| 10                  | Hold       |         |
appendix g laboratory methodology for
exo-sap-it protocol

1) Add 2-4 uL of ExoSap-It enzyme reagent to each PCR well. 2) Perform the reaction using a thermocycler under the following conditions:
appendix g laboratory methodology for
iii. abi sequencing reaction (see

1) In 96 well reaction trays, add 2 μL each of the PCR amplicons into the appropriate wells. 2) Spin down the plates to get rid of bubbles. 3) Add 8 uL of sequencing mix as noted below.  
| 37   | 30 min   |
|------|----------|
| 80   | 15 min   |
| 10   | hold     |  
(uL) 120 1.2 5 X 0.6 X Big Dye Sequencing Buffer (uL) 32 0.32 10 uM 320 nM M13 for OR M13 Rev primer,  
0 2 ExoSAP- treated PCR product  
(uL) 488 4.88 H2O  
| n=   | 100   | n=1   | 0   | Unit   | Final   |    |
|------|-------|-------|-----|--------|---------|----|
| (uL) | 160   | 1.6   | 2.5 | X      | 0.4     | X  |
| (uL) | 488   | 4.88  | H2O |        |         |    |  
4) Cover and place in the DNA Engine Tetrad / PTC-225 Peltier Thermal Cycler and select the program "m13seq" using the following conditions:  
| Cycle Sequencing   | m13seq   |        |
|--------------------|----------|--------|
|                    | 96       | 1 min  |
| 25 cycles          | 96       | 10 sec |
|                    | 50       | 5 sec  |
|                    | 60       | 4 min  |
|                    | 10       | Hold   |  
Label a new set of 96 well plates before proceeding to the Qiagen DyeEx 96 Kit Protocol.
appendix g laboratory methodology for
iv. sequencing reaction purification

Protocol: The sequencing reactions can be purified using the BigDye XTerminator Kit from Applied Biosystems.  
1. Add 45μL of SAM solution directly to each sequencing reaction well (heat SAM solution to 37°C if precipitates are present).  
2. Vortex well to resuspend BigDye Xterminator Beads and add 10μL to each reaction well. 3. Seal plate with foil and vortex at 1800rpm for 30 minutes on an Illumina High-Speed Microplate Shaker.  
4. Centrifuge the plate at 1000 xg for 2 minutes. 5. The plate is now ready to be loaded onto the 3730 DNA Analyzer. Check that the instrument protocol is set to BDx-FastSeq50_Pop7_v3_Z to ensure proper capillary height during injection.
appendix g laboratory methodology for
v. quick guide to applied

1. Turn on the 3730 DNA Analyzer instrument. 2. Log into the Applied Biosystems computer that is connected to the 3730 DNA Analyzer hardware using the following information:
Username: Administrator Password: MeltzerLab 3. Run Applied Biosystems Software (Foundations 3730 Data Collection program).  
Go to Plate Manager and enter File name. The file name should include: [Gene]_[gene section or region]_[sample ID]_[PI] _[plate ID]_[F or R]_[date run] (denoting F for forward primer and R for reverse primer).  
4. At Run Scheduler add the files in the order in which you would like the plates to be run.  
The bottom plate will be run first.  
5. Clean out all trays labeled Buffer, *Water*, and *Waste* by rinsing with water. Refill deionized water up to the black lines in the *Water* and *Waste* trays. Add 1X 3730 Applied 87 Biosystems buffer (after diluting original 10X concentration) into tray labeled *Buffer*. Place tray back into the 3730 DNA Analyzer.  
6. If bubbles are present in the capillary tubes, you MUST run Bubble Wizard to remove bubbles. Sample plates must be loaded in the In tray to run this program. If a bubble is present and the Bubble Wizard is not run, the capillary tubes can be damaged.  
7. Change Sequencing Buffer in small glass jar next to the DNA polymer and fill the jar to the red line.  
8. Close doors, and turn off lights (if turned on) by pushing the button with the light bulb on the left side of the 3730 DNA Analyzer instrument. The light should be green on the outside of the 3730 DNA Analyzer. If the computer is ready to run, a green *Play* button should be highlighted on the computer in the program under Run Scheduler. Click to run.  
9. Discard plates after they have been run.
appendix g laboratory methodology for
vi. dna analysis using variant

1. Open and import all files. 2. Import the associated reference sequence file. 3. Click '*Analyze button*.' 4. Click on Variant Review tab for visual chromatogram analysis of all samples. 5. Save sequencing project in the following format: PI_CMPC study ID_Gene name_date of sequencing.
appendix g laboratory methodology for
vii. resulting reads

Ultimately, the scoring is the judgment of the technician and director reviewing the results. There are standard quality control measures, but we only read sequence that is well above the level of these cutoffs - in other words, we are reading in the best part of reads. Generally, we strive for bidirectional reads. On those occasions when the complexity of the sequence on one end is just so low that getting one of the directions to work is really difficult, but the other strand can be sequenced quite well, a judgment call will be made by the CLIA director as to whether to accept the current results, to rerun the sample, or report out with a caveat that the results are suspect. A failed read needs to be repeated if that exon has been shown in other cancer patients to contain a possible mutation. Questionable reads should also be repeated if there is a known hotspot. Instead of "questionable" we will use the term "lower quality" or LQ. We will rely upon the software to correctly call all negative bases but a few spot checks (~6) will be performed by the technician as a QC measure. However, the CLIA laboratory director or his designate will visually review any mutations prior to reporting the results to the clinician. In general, we will not do validation of mutations; that can be kicked back to the investigator. For some projects, validation might be important, but due to limited resources we will discuss on a case by case basis, first internally, and then with the investigator, if appropriate. In general, good quality bidirectional sequence is definitive. Result reporting nomenclature will be guided by the Update on Nomenclature for Human Gene Mutations, Ad Hoc Committee on Mutation Nomenclatures1.
appendix g laboratory methodology for
viii. quality control pcr quality

QC data tracking will be documented via the PCR Amplicon Quality Control Data Tracking form.  
1. **Positive controls:** No external positive control (PC) is necessary for this assay, rather the amplification of the patient DNA is proof that the PCR reagents and method are functioning correctly.  
2. **Negative controls:** No template controls (NTC) should be run for each primer set used in the assay for each run. These are to control against contamination of the reagents or reaction environment from foreign DNA or amplicons. A positive NTC
rules out against the use of that primer reaction in the assay and must subsequently be repeated after making use of fresh reagents and cleaning the work area with 10% bleach.  
3. **Gel electrophoresis:** All NTCs and a random sampling of the patient amplicons will be run on an agarose gel to show that there is no contamination and to confirm that the reagents and PCR method is working as expected.  
Acceptable limits: no band for NTCs and visible bands of the expected size for patient amplicons. Results will not be reported out if controls do not pass QC.
appendix g laboratory methodology for
dna sequencing quality control

The *Quality Number* generated by Variant Reporter is a numerical scale (ranging from 0-99) that is used in order to compare the quality of the sample sequence with the GenBank reference sequence. Generally, the Quality Number for the sample sequence should be 5 or above in order to make an accurate comparison with the GenBank reference sequence. However, sometimes a case may occur where the forward or reverse sequence may be of low quality, while its corresponding forward or reverse sequence may be above a quality number of 5 and satisfactory. In this case, use the higher quality sequence to compare with the reference sequence. Every so often, a region of the higher quality sequence may be unreadable. When this occurs, look at the same region of the sequence in the reverse direction. If that region is clear, then review that particular region and continue analysis, but if that region is also unreadable then that sample sequence should be marked as *Failure*. Ultimately, however, the Quality Number is only a guide and the final determination of sequence quality is the technician's. The test read should also be compared to the normal sequence generated during the validation stage of the test.
appendix g laboratory methodology for
ix. troubleshooting and corrective actions

1. If plate is loaded incorrectly into the ABI 3700, look for two problems: (1) Is the white lid snapped tightly onto the plate holder (2) Are the capillary needles still straight? Solution (1) is to readjust the lid for the plate holder. Solution (2) is to shut off the ABI 3700 and seek technical support to readjust capillary needles.  
2. If error has occurred overnight then fix the error and either run same plate again or rerun Dye-Ex clean up using the saved plate from the sequencing reaction.
appendix g laboratory methodology for
x. references

1. Cotton, RGH. *Update on Nomenclature for Human Gene Mutations*. Human Mutation
(1996), 8:197-202.  
2. Patrick C. Ma, Maria S. Tretiakova, Alexander C. MacKinnon, Nithya Ramnath, Candace Johnson, Sascha Dietrich, Tanguy Seiwert, James G. Christensen, Ramasamy Jagadeeswaran, Thomas Krausz, Everett E. Vokes, Aliya N. Husain, Ravi Salgia, Expression and mutational analysis of MET in human solid cancers. Genes, Chromosomes and Cancer (2008), 47 (12); 1025-1037.  
3. *Fluorescent DNA Sequencing on the ABI 3730/3730xl*. Mayo Clinic: Department of Laboratory Medicine and Pathology Molecular Genetics Laboratory, Rochester, MN 55905.  
4. Fluorescence-based Dideoxy Sequencing - ABI Prism BigDye Terminator Cycle Sequencing Kit. Division of Laboratory Medicine, Clinical Center, NIH, Bethesda 20892  
| SOP Revision Tracking Sheet                                                 |     |                       |          |
|-----------------------------------------------------------------------------|-----|-----------------------|----------|
| Prepared by: Robert Chang, BS, and Marbin Pineda, MS, Daniel C Edelman, PhD |     |                       |          |
| Date                                                                        | By  | Signature             | Comments |
| Jonathan K. Killian, MD,                                                    |     |                       |          |
| Adopted                                                                     | PhD | Original SOP for CLIA |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| ■Reviewed ■Revised                                                          |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| ■Reviewed ■Revised                                                          |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| - Reviewed - Revised                                                        |     |                       |          |
| Discontinued:                                                               |     |                       |          |
dna pcr and sequencing assays
appendix j primers for cdkn2a

| Forward  primer -  Relative  position †   | Forward primer sequence§   | Reverse  primer -  Relative  position ‡   | Reverse primer sequence§   |     |                               |
|-------------------------------------------|----------------------------|-------------------------------------------|----------------------------|-----|-------------------------------|
| Exon #                                    | Genomic position           | M13-                                      |                            |     |                               |
| 1                                         | chr9:21964676-21964826     | -114                                      | AGCACCGGAGGAAGAAAGAG       | 66  | AGCGCTACCTGATTCCAATTC         |
| M13-                                      |                            |                                           |                            |     |                               |
| 2                                         | chr9:21960900-21961207     | -73                                       | GGGCTCTACACAAGCTTCCTTTC    | 67  | CCGGGCTGAACTTTCTGTG           |
| 3                                         | chr9:21958227-21958241     | -294                                      | TCTTCTTGCAACCCTGCG         | 59  | M13-GGCAGTTGTGGCCCTGTAG  M13- |
| 1                                         | chr9:21984137-21984453     | -101                                      | ATCTTGGAGGTCCGGGTG         | 69  | CCTGGGCTAGAGACGAATTATC        |
| M13-                                      |                            |                                           |                            |     |                               |
| 2                                         | chr9:21961001-21961207     | -72                                       | GGCTCTACACAAGCTTCCTTTC     | 60  | CTATGCGGGCATGGTTACTG          |
| M13-                                      |                            |                                           |                            |     |                               |
| 1                                         | chr17:7520563-7520637      | -74                                       | AGGGTTGGAAGTGTCTCATGC      | 139 | AGCCCAACCCTTGTCCTTAC          |
| M13-                                      |                            |                                           |                            |     |                               |
| 2                                         | chr17:7520424-7520446      | -179                                      | CAGTCAGATCCTAGCGTCGAG      | 120 | AAATCATCCATTGCTTGGGAC  M13-   |
| 3                                         | chr17:7520036-7520315      | -117                                      | ACGTTCTGGTAAGGACAAGGG      | 69  | GAGGAATCCCAAAGTTCCAAAC  M13-  |
| 4                                         | chr17:7519095-7519279      | -154                                      | AAGCTCCTGAGGTGTAGACGC      | 69  | GGGCCAGACCTAAGAGCAATC         |
| 5                                         | chr17:7518901-7519014      | -98                                       | M13-CTGCTCAGATAGCGATGGTG   | 169 | AGGCCCTTAGCCTCTGTAAGC  M13-   |
| 6                                         | chr17:7518223-7518333      | -286                                      | CATCCTGGCTAACGGTGAAAC      | 70  | AGAAATCGGTAAGAGGTGGGC  M13-   |
| 7                                         | chr17:7517743-7517880      | -92                                       | GTTGGGAGTAGATGGAGCCTG      | 81  | TTGGGCAGTGCTAGGAAAGAG         |
| M13-                                      |                            |                                           |                            |     |                               |
| 8                                         | chr17:7517577-7517651      | -107                                      | GGAGCACTAAGCGAGGTAAGC      | 110 | TTGTCTTTGAGGCATCACTGC  M13-   |
| 9                                         | chr17:7514651-7514758      | -222                                      | ATTGCACCATTGCACTCCC        | 81  | AGCTGCCTTTGACCATGAAG          |
| 10                                        | chr17:7513651-7513733      | -124                                      | M13-CCATCTTGATTTGAATTCCCG  | 76  | ATTGCAAGCAAGGGTTCAAAG         |
appendix k guidelines for management
appendix j primers for cdkn2a

If GI adverse events are not appropriately managed, they may be associated with the development of dehydration. Management of gastrointestinal adverse events is discussed in detail below.
appendix k guidelines for management
nausea vomiting or both

In subjects who have emesis and are unable to retain lapatinib, every attempt should be made to obtain control of nausea and vomiting. A dose may be repeated if tablets can be visually found after the vomiting episode.
appendix k guidelines for management
diarrhea

These broad general management principles are recommended to proactively try and avoid more serious complications by active management of diarrhea syndrome. Guidelines such as these should never replace sound clinical judgment. Experience thus far suggests that when lapatinib is used as monotherapy, uncomplicated Grade 1 or 2 diarrhea is most prevalent. These general management principles do not address comprehensive management of more serious or protracted diarrhea syndromes. Common clinical sense with the onset of uncomplicated Grade 1-2 diarrhea: stop all lactose containing products: drink 8-10 large glasses of clear liquids a day; eat frequent small meals; for Grade 2 diarrhea hold cytotoxic chemotherapy, and consider a dose reduction of lapatinib (discuss with Sponsor); administer standard doses of loperamide: initial dose 4 mg followed by 2 mg every 4 hours or after every unformed stool. It is suggested to continue loperamide until the subject is free from diarrhea for 12 hours. For Grade 3 or 4 diarrhea or Grade 1 or 2 with complicating features (severe cramping, severe nausea/vomiting, decreased performance status, fever, sepsis, Grade 3 or 4 neutropenia, frank bleeding, dehydration) use intravenous fluids as appropriate, consider hospital administration. Use prophylactic antibiotics as needed (example fluoroquinolones) especially if diarrhea is persistent beyond 24 hours or there is a fever or Grade 3-4 neutropenia, hold both cytotoxic chemotherapy and lapatinib and discuss with medical monitor.
appendix k guidelines for management
treatment of gastrointestinal adverse events

Diarrhea can be debilitating, and on rare occasions, it is potentially life-threatening. Based on experience with lapatinib alone or in combination with taxanes and/or trastuzumab, diarrhea should be managed proactively to avoid complications or worsening of the patient's condition. Guidelines developed by an American Society of Clinical Oncology (ASCO) panel for treating chemotherapy-induced diarrhea are abstracted below. Pharmacological approaches include the following: Loperamide, administered as an initial 4-mg dose, followed by 2-mg doses every 4 hours. This dose and regimen are moderately effective. Clonidine, non-steroidal anti-inflammatory drugs, and the serotonin antagonist cyproheptadine have been shown to be effective in controlling diarrhea associated with inflammation of the bowel. The synthetic octapeptide, octreotide, has been shown to be effective in the control of diarrhea induced by fluoropyrimidine-based chemotherapy regimens when administered as an escalating dose by continuous infusion or subcutaneous injection. Octreotide can be administered at doses ranging from 100 μg twice daily to 500 μg 3 times daily, with a maximum-tolerated dose of 2000 μg 3 times daily in a 5-day regimen.
appendix l ctep multicenter guidelines
treatment of gastrointestinal adverse events

If an institution wishes to collaborate with other participating institutions in performing a CTEP sponsored research protocol, then the following guidelines must be followed.
appendix l ctep multicenter guidelines
responsibility of the protocol chair

The Protocol Chair will be the single liaison with the CTEP Protocol and Information Office (PIO). The Protocol Chair is responsible for the coordination, development, submission, and approval of the protocol as well as its subsequent amendments. The protocol must not be rewritten or modified by anyone other than the Protocol Chair. There will be only one version of the protocol, and each participating institution will use that document. The Protocol Chair is responsible for assuring that all participating institutions are using the correct version of the protocol.  
The Protocol Chair is responsible for the overall conduct of the study at all participating institutions and for monitoring its progress. All reporting requirements to CTEP are the responsibility of the Protocol Chair. The Protocol Chair is responsible for the timely review of Adverse Events (AE) to assure safety of the patients. The Protocol Chair will be responsible for the review of and timely submission of data for study analysis.  
Responsibilities of the Coordinating Center Each participating institution will have an appropriate assurance on file with the Office for Human Research Protection (OHRP), NIH. The Coordinating Center is responsible for assuring that each participating institution has an OHRP assurance and must maintain copies of IRB approvals from each participating site. Prior to the activation of the protocol at each participating institution, an OHRP form 310 (documentation of IRB approval) must be submitted to the CTEP PIO. The Coordinating Center is responsible for central patient registration. The Coordinating Center is responsible for assuring that IRB approval has been obtained at each participating site prior to the first patient registration from that site. The Coordinating Center is responsible for the preparation of all submitted data for review by the Protocol Chair. The Coordinating Center will maintain documentation of AE reports. There are two options for AE reporting: (1) participating institutions may report directly to CTEP with a copy to the Coordinating Center, or (2) participating institutions report to the Coordinating Center who in turn report to CTEP. The Coordinating Center will submit AE reports to the Protocol Chair for timely review. Audits may be accomplished in one of two ways: (1) source documents and research records for selected patients are brought from participating sites to the Coordinating Center for audit, or (2) selected patient records may be audited on-site at participating sites. If the NCI chooses to have an audit at the Coordinating Center, then the Coordinating Center is responsible for having all source documents, research records, all IRB approval documents, NCI Drug Accountability Record forms, patient registration 95 lists, response assessments scans, x-rays, etc. available for the audit.
appendix l ctep multicenter guidelines
inclusion of multicenter guidelines in

The protocol must include the following minimum information:
¾ The title page must include the name and address of each participating institution and the name, telephone number and e-mail address of the responsible investigator at each participating institution.  
¾ The Coordinating Center must be designated on the title page. ¾ Central registration of patients is required. The procedures for registration must be stated in the protocol.  
¾ Data collection forms should be of a common format. Sample forms should be submitted with the protocol. The frequency and timing of data submission forms to the Coordinating Center should be stated.  
¾ Describe how AEs will be reported from the participating institutions, either directly to CTEP or through the Coordinating Center.  
¾ Describe how Safety Reports and Action Letters from CTEP will be distributed to participating institutions.
appendix l ctep multicenter guidelines
agent ordering

Except in very unusual circumstances, each participating institution will order DCTDsupplied agents directly from CTEP. Agents may be ordered by a participating site only after the initial IRB approval for the site has been forwarded by the Coordinating Center to the CTEP PIO.