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10.2.1 sample processing

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Genomic DNA from histologically characterized tissue will be sequenced at loci encoding each candidate gene. DNA will be amplified using a PCR device and using exon specific primers. The PCR products will then be pooled and sequenced directly using a next generation sequencing device (i.e. Illumina Genome Analyzer). This will allow the parallel sequencing of the entire panel of candidate genes with rapid turnaround. 10.2.2 Gene sequencing Somatic gene mutation analysis including but not limited to the following list of genes will be performed in the available tumor samples:  
| Version Date: 01/07/2020 Approved  Approved Gene   | Symbol                                                                    | Sequence                                            | Previous      | Aliases         |                               |
|----------------------------------------------------|---------------------------------------------------------------------------|-----------------------------------------------------|---------------|-----------------|-------------------------------|
| Gene                                               | Name                                                                      | Location                                            | Accession IDs | Symbols         |                               |
| AKT1                                               | v-akt murine thymoma  viral oncogene homolog  1                           | 14q32.32-                                           | M63167        | RAC, PKB,       |                               |
| q32.33                                             | NM_005163                                                                 | PRKBA, AKT                                          |               |                 |                               |
| AKT2                                               | v-akt murine thymoma  viral oncogene homolog  2                           | 19q13.1-                                            | NM_001626     |                 |                               |
| q13.2                                              |                                                                           |                                                     |               |                 |                               |
| V                                                  |                                                                           |                                                     |               |                 |                               |
| ALK                                                | anaplastic lymphoma                                                       | 2q23                                                | D45915        | CD246           |                               |
| receptor tyrosine kinase                           | NM_004304                                                                 |                                                     |               |                 |                               |
| APC                                                | adenomatous polyposis                                                     | 5q21-q22                                            | M74088        | DP2, DP3, DP2.5 |                               |
| coli                                               | NM_000038                                                                 |                                                     |               |                 |                               |
| ART                                                | anaplastic lymphoma                                                       | 2p23                                                | D45915        | CD246           |                               |
| receptor tyrosine kinase                           | NM_004304                                                                 |                                                     |               |                 |                               |
| ATM                                                | ataxia telangiectasia                                                     | 11q22-q23                                           | AB209133      | ATA, ATDC,      | TEL1, TELO1                   |
| mutated                                            | NM_000051                                                                 | ATC, ATD                                            |               |                 |                               |
| O                                                  |                                                                           |                                                     |               |                 |                               |
| BCL2                                               | B-cell CLL/lymphoma                                                       | 18q21.3                                             | M14745        |                 |                               |
| 2                                                  | NM_000633,  NM_000657                                                     | Bcl-2                                               |               |                 |                               |
| BMPR1A                                             | bone morphogenetic  protein receptor, type  IA                            | 10q22.3                                             | BC028383      | ACVRLK3         | ALK3, CD292                   |
| NM_004329                                          |                                                                           |                                                     |               |                 |                               |
| BMPR1B                                             | bone morphogenetic  protein receptor, type  IB                            | 4q23-q24                                            | D89675        | ALK6, CDw293    |                               |
| NM_001203                                          |                                                                           |                                                     |               |                 |                               |
| I                                                  |                                                                           |                                                     |               |                 |                               |
| BMPR2                                              | bone morphogenetic  protein receptor, type II  (serine/threonine  kinase) | 2q33-q34                                            | Z48923        | PPH1            | BRK-3, T-ALK,  BMPR3, BMPR-II |
| BRAF                                               | v-raf murine sarcoma  viral oncogene homolog  B1                          | 7q34                                                | M95712        | BRAF1           |                               |
| NM_004333                                          |                                                                           |                                                     |               |                 |                               |
| CDKN2A                                             | cyclin-dependent kinase  inhibitor 2A  (melanoma, p16,  inhibits CDK4)    | 9p21                                                | L27211        | CDKN2,          | CDK4I, p16,                   |
| NM_000077                                          | MLM                                                                       | INK4a, MTS1,  CMM2, ARF, p19,  p14, INK4,  p16INK4a |               |                 |                               |
| CDKN2B                                             | cyclin-dependent kinase  inhibitor 2B (p15,  inhibits CDK4)               | D                                                   |               |                 |                               |
| 9p21                                               | AB060808                                                                  | P15, MTS2,                                          |               |                 |                               |
| NM_004936                                          | INK4B, TP15,  CDK4I, p15INK4b                                             |                                                     |               |                 |                               |
| CDKN2C                                             | cyclin-dependent kinase  inhibitor 2C (p18,  inhibits CDK4)               | 1p32.3                                              | BC000598      | INK4C, p18      |                               |
| NM_001262                                          |                                                                           |                                                     |               |                 |                               |  
| Version Date: 01/07/2020 CDH1 cadherin 1, type 1, Ecadherin (epithelial)                           | 16q22.1                                                                                                             | L08599                | UVO                  | uvomorulin, CD324   |                 |
|---------------------------|---------------------------------------------------------------------------------------------------------------------|-----------------------|----------------------|---------------------|-----------------|
| NM_004360                 |                                                                                                                     |                       |                      |                     |                 |
| CTNNB1                    | catenin (cadherinassociated protein), beta  1, 88kDa                                                                                                                     | 3p21                  | X87838               | CTNNB               | beta-catenin    |
| NM_001098210              |                                                                                                                     |                       |                      |                     |                 |
| EGFR                      | epidermal growth factor  receptor (erythroblastic  leukemia viral (v-erb-b)  oncogene homolog,  avian)              | 7p12                  | NM_005228            | ERBB                | ERBB1           |
| V                         |                                                                                                                     |                       |                      |                     |                 |
| EML4                      | echinoderm microtubule                                                                                              | 2p21                  | AF177377             | C2orf2              | ROPP120, ELP120 |
| associated protein like 4 | NM_019063                                                                                                           |                       |                      |                     |                 |
| EPHA3                     | EPH receptor A3                                                                                                     | 3p11.2                | M83941               | ETK, ETK1,          | HEK, HEK4       |
| NM_005233                 | TYRO4                                                                                                               |                       |                      |                     |                 |
| EPHA5                     | EPH receptor A5                                                                                                     | 4q13.1                | L36644               | Hek7, TYRO4,        |                 |
| NM_004439                 | CEK7, EHK1                                                                                                          |                       |                      |                     |                 |
| O                         |                                                                                                                     |                       |                      |                     |                 |
| EPHA7                     | EPH receptor A7                                                                                                     | 6q16.3                | L36642               | Hek11               |                 |
| ERBB2                     | v-erb-b2 erythroblastic  leukemia viral  oncogene homolog 2,  neuro/glioblastoma  derived oncogene  homolog (avian) | 17q11.2-              | X03363               | NGL                 | NEU, HER-2,     |
| q12                       | CD340, HER2                                                                                                         |                       |                      |                     |                 |
| ERBB3                     | v-erb-b2 erythroblastic  leukemia viral  oncogene homolog 3  (avian)                                                | 12q13                 | M34309               | LCCS2               | HER3            |
| I                         |                                                                                                                     |                       |                      |                     |                 |
| ERBB4                     | v-erb-a erythroblastic  leukemia viral  oncogene homolog 4  (avian)                                                 | 2q33.3-q34            | L07868  NM_001042599 |                     |                 |
| FAS                       | Fas (TNF receptor                                                                                                   | 10q24.1               | M67454               | FAS1, APT1,         | CD95, APO-1     |
| superfamily, member 6)    | TNFRSF6                                                                                                             |                       |                      |                     |                 |
| FGFR1                     | fibroblast growth factor                                                                                            | 8p12                  | M34185               | FLT2, KAL2          | H2, H3, H4, H5, |
| receptor 1                | CEK, FLG,  BFGFR, N-SAM,  CD331                                                                                     |                       |                      |                     |                 |
| D                         |                                                                                                                     |                       |                      |                     |                 |
| FGFR2                     | fibroblast growth factor                                                                                            | 10q25.3-              | AK026508             |                     |                 |
| receptor 2                | q26                                                                                                                 | NM_022976,  NM_000141 | KGFR, BEK,           | CEK3, TK14,         |                 |
| CFD1, JWS                 | TK25, ECT1, KSAM, CD332                                                                                                                     |                       |                      |                     |                 |
| FGFR3                     | fibroblast growth factor                                                                                            | 4p16.3                | M64347               | ACH                 | CEK2, JTK4,     |
| receptor 3                | CD333                                                                                                               |                       |                      |                     |                 |
| FGFR4                     | fibroblast growth factor                                                                                            | 5q33-qter             | AF202063             | JTK2, CD334         |                 |
| receptor 4                |                                                                                                                     |                       |                      |                     |                 |
| FLT4                      | fms-related tyrosine                                                                                                | 5q34-q35              | X68203               | VEGFR3, PCL         |                 |
| kinase 4                  | 100                                                                                                                 |                       |                      |                     |                 |  
| Version Date: 01/07/2020 FZD1 frizzled homolog 1   | 7q21                                                                                       | AB017363               | DKFZp564G072   |                             |                |
|----------------------------------------------------|--------------------------------------------------------------------------------------------|------------------------|----------------|-----------------------------|----------------|
| (Drosophila)                                       | NM_003505                                                                                  |                        |                |                             |                |
| GNAS                                               | GNAS complex locus                                                                         | 20q13.2-               | M21142         | GNAS1                       | NESP55, NESP,  |
| q13.3                                              | NM_000516                                                                                  | GNASXL, GPSA,  SCG6    |                |                             |                |
| GSK3B                                              | glycogen synthase                                                                          | 3q13.3                 | BC012760       |                             |                |
| kinase 3 beta                                      |                                                                                            |                        |                |                             |                |
| V                                                  |                                                                                            |                        |                |                             |                |
| HIF1A                                              | hypoxia inducible factor  1, alpha subunit (basic  helix-loop-helix  transcription factor) | 14q23.2                | U22431         | MOP1, HIF-1alpha,           |                |
| NM_001530                                          | PASD8, HIF1,  bHLHe78                                                                      |                        |                |                             |                |
| HRAS                                               | v-Ha-ras Harvey rat  sarcoma viral oncogene  homolog                                       | 11p15.5                | AJ437024       | HRAS1                       |                |
| NM_176795                                          |                                                                                            |                        |                |                             |                |
| IGF1R                                              | insulin-like growth                                                                        | 15q26.3                | M69229         | JTK13, CD221,               |                |
| factor 1 receptor                                  | NM_000875                                                                                  | IGFIR, MGC18216,  IGFR |                |                             |                |
| O                                                  |                                                                                            |                        |                |                             |                |
| INHBA                                              | inhibin, beta A                                                                            | 7p15-p13               |                |                             |                |
| KDR                                                | kinase insert domain  receptor (a type III  receptor tyrosine  kinase)                     | 4q11-q12               | AF035121       | FLK1, VEGFR,  VEGFR2, CD309 |                |
| KIT                                                | v-kit Hardy-Zuckerman  4 feline sarcoma viral  oncogene homolog                            | 4q11-q12               | S67773         | PBT                         | CD117, SCFR, CKit                |
| I                                                  |                                                                                            |                        |                |                             |                |
| KRAS                                               | v-Ki-ras2 Kirsten rat  sarcoma viral oncogene  homolog                                     | 12p12.1                | BC010502       | KRAS2                       | KRAS1          |
| NM_033360                                          |                                                                                            |                        |                |                             |                |
| LRP1B                                              | low density lipoproteinrelated protein 1B  (deleted in tumors)                                                                                            | 2q21.2                 | AF176832       | LRP-DIT                     |                |
| NM_018557                                          |                                                                                            |                        |                |                             |                |
| LTK                                                | leukocyte receptor                                                                         | 15q15.1-               | D16105         | TYK1                        |                |
| tyrosine kinase                                    | q21.1                                                                                      | D                      |                |                             |                |
| MAPK1                                              | mitogen-activated                                                                          | 22q11.2                | M84489         | PRKM2,                      | ERK, ERK2,     |
| protein kinase 1                                   | PRKM1                                                                                      | p41mapk, MAPK2         |                |                             |                |
| MAPK3                                              | mitogen-activated                                                                          | 16p11.2                | M84490         | PRKM3                       | ERK1, p44mapk, |
| protein kinase 3                                   | p44erk1                                                                                    |                        |                |                             |                |
| MAP2K1                                             | mitogen-activated                                                                          | 15q22.1-               | L11284         | PRKMK1                      | MEK1, MAPKK1   |
| protein kinase kinase 1                            | q22.33                                                                                     |                        |                |                             |                |  
| Version Date: 01/07/2020 MAP2K2 mitogen-activated   | 19p13.3                                                      | L11285                  | PRKMK2             | MEK2                                     |                |
|-----------------------------------------------------|--------------------------------------------------------------|-------------------------|--------------------|------------------------------------------|----------------|
| protein kinase kinase 2                             |                                                              |                         |                    |                                          |                |
| MET                                                 | met proto-oncogene  (hepatocyte growth  factor receptor)     | 7q31                    | M35073             | HGFR, RCCP2                              |                |
| mTOR                                                | mechanistic target of  rapamycin  (serine/threonine  kinase) | 1p36                    | L34075             | FRAP,                                    |                |
| NM_004958                                           | FRAP2,  FRAP1                                                | RAFT1, RAPT1,  FLJ44809 |                    |                                          |                |
| V                                                   |                                                              |                         |                    |                                          |                |
| NF1                                                 | neurofibromin 1                                              | 17q11.2                 | NM_000267          |                                          |                |
| NOTCH1                                              | Notch homolog 1,  translocation-associated  (Drosophila)     | 9q34.3                  | AF308602           | TAN1                                     |                |
| NM_017617                                           |                                                              |                         |                    |                                          |                |
| NRAS                                                | neuroblastoma RAS  viral (v-ras) oncogene  homolog           | 1p13.2                  | BC005219 NM_002524 |                                          |                |
| NTRK1                                               | neurotrophic tyrosine                                        | 1q21-q22                | Y09028             | TRK, TRKA, MTC                           |                |
| kinase, receptor, type 1                            | NM_002529                                                    |                         |                    |                                          |                |
| O                                                   |                                                              |                         |                    |                                          |                |
| NTRK2                                               | neurotrophic tyrosine                                        | 9q22.1                  | AF410902           | TRKB                                     |                |
| kinase, receptor, type 2                            |                                                              |                         |                    |                                          |                |
| NTRK3                                               | neurotrophic tyrosine                                        | 15q24-q25               | U05012             | TRKC                                     |                |
| kinase, receptor, type 3                            |                                                              |                         |                    |                                          |                |
| PAK3                                                | p21 protein  (Cdc42/Rac)-activated  kinase 3                 | Xq22.3                  | AF068864           | MRX30,                                   | hPAK3, bPAK    |
| NM_002578                                           | MRX47                                                        |                         |                    |                                          |                |
| PDGFRA                                              | platelet-derived growth  factor receptor, alpha  polypeptide | 4q12                    | D50001             | CD140a, PDGFR2                           |                |
| NM_006206                                           |                                                              |                         |                    |                                          |                |
| I                                                   |                                                              |                         |                    |                                          |                |
| PIK3CA                                              | phosphoinositide-3- kinase, catalytic, alpha  polypeptide    | 3q26.3                  | NM_006218          | p110-alpha,  MGC142161, PI3K,  MGC142163 |                |
| PLK1                                                | polo-like kinase 1                                           | 16p                     | NM_005030          | PLK                                      |                |
| (Drosophila)                                        |                                                              |                         |                    |                                          |                |
| PRKDC                                               | protein kinase, DNAactivated, catalytic  polypeptide                                                              | 8q11                    | NM_001081640       | HYRC,                                    | DNPK1, p350,   |
| HYRC1                                               | DNAPK, XRCC7,  DNA-PKcs                                      |                         |                    |                                          |                |
| PRKCG                                               | protein kinase C,                                            | 19q13.4                 | M13977             | PKCG,                                    | PKCC, MGC57564 |
| gamma                                               | NM_002739                                                    | SCA14 D                 |                    |                                          |                |
| PTCH1                                               | patched homolog 1                                            | 9q22.1-q31              | AI494442           | NBCCS,                                   | BCNS           |
| (Drosophila)                                        | NM_000264                                                    | PTCH                    |                    |                                          |                |
| PTEN                                                | phosphatase and tensin                                       | 10q23                   | U92436             | BZS,                                     | MMAC1, TEP1,   |
| homolog                                             | NM_000314                                                    | MHAM                    | PTEN1              |                                          |                |
| PTPRD                                               | protein tyrosine  phosphatase, receptor  type, D             | 9p24.1-p23              | X54133             | PTPD, HPTP                               |                |  
| Version Date: 01/07/2020 RB1 retinoblastoma 1   | 13q14.2                                                                    | M15400   | OSRC     | RB             |                          |
|-------------------------------------------------|----------------------------------------------------------------------------|----------|----------|----------------|--------------------------|
| RHOA                                            | ras homolog gene                                                           | 3p21.3   | BC001360 | ARH12,         | RhoA, Rho12,             |
| family, member A                                | NM_001664                                                                  | ARHA     | RHOH12   |                |                          |
| SMAD2                                           | SMAD family member                                                         | 18q21    | U65019   | MADH2          | MADR2, JV18-1            |
| 2                                               | NM_005901                                                                  |          |          |                |                          |
| SMAD4                                           | SMAD family member                                                         | 18q21.1  | U44378   | MADH4          | DPC4                     |
| 4                                               | NM_005359                                                                  |          |          |                |                          |
| V                                               |                                                                            |          |          |                |                          |
| SLC38A3                                         | solute carrier family 38,                                                  | 3p21.3   | U49082   | G17, SN1       |                          |
| member 3                                        | NM_006841                                                                  |          |          |                |                          |
| SMG1                                            | SMG1 homolog,  phosphatidylinositol 3- kinase-related kinase  (C. elegans) | 16p12.3  | AB061371 | LIP, KIAA0421, |                          |
| NM_015092                                       | ATX                                                                        |          |          |                |                          |
| SMO                                             | smoothened homolog                                                         | 7q32.1   | U84401   | SMOH           |                          |
| (Drosophila)                                    | NM_005631                                                                  |          |          |                |                          |
| STK11                                           | serine/threonine kinase                                                    | 19p13.3  | U63333   | PJS, LKB1      |                          |
| 11                                              | NM_000455                                                                  |          |          |                |                          |
| O                                               |                                                                            |          |          |                |                          |
| STK33                                           | serine/threonine kinase                                                    | 11p15.3  | AJ303380 |                |                          |
| 33                                              | NM_030906                                                                  |          |          |                |                          |
| TGFB1                                           | transforming growth                                                        | 19q13.1  | X02812   | TGFB,          | CED, TGFbeta             |
| factor, beta 1                                  | DPD1                                                                       |          |          |                |                          |
| TGFB2                                           | transforming growth                                                        | 1q41     | M19154   |                |                          |
| factor, beta 2                                  | NM_003238                                                                  |          |          |                |                          |
| TP53                                            | tumor protein p53                                                          | 17p13.1  | AF307851 | p53, LFS1      |                          |
| NM_000546                                       |                                                                            |          |          |                |                          |
| I                                               |                                                                            |          |          |                |                          |
| TSC1                                            | tuberous sclerosis 1                                                       | 9q34     | AF013168 | TSC            | KIAA0243, LAM,  hamartin |
| TSC2                                            | tuberous sclerosis 2                                                       | 16p13.3  | AB014460 | TSC4           | tuberin, LAM             |
| NM_000548                                       |                                                                            |          |          |                |                          |
| Wnt                                             | wingless-type MMTV  integration site family,  member 1                     | 12q13    | X03072   | INT1 D         |                          |
| ZMYND10                                         | zinc finger, MYNDtype containing 10                                                                            | 3p21.3   | U70824   | BLU            |                          |
| NM_015896                                       |                                                                            |          |          |                |                          |
| 10.3                                            | Other molecular profiling analyses in tumor samples                        |          |          |                |                          |  
If enough material is available, other molecular profiling analyses including but not limited to the following will be performed:  
1) Comparative genomic hybridization (CGH) 2) FISH analysis of candidate genes 3) Methylation analysis 4) MicroRNAs 5) Gene expression arrays 6) Full tumor genome sequencing
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10.4 non-invasive biomarkers studies (only

V
Biomarkers, including prognostic and predictive factors, are widely studied in oncology and are important in anticancer drug development (112). As mentioned above, recurrent somatic gene mutations, such as EGFR, KRAS, p53, and LKB-1, have been detected in lung cancers (113).  
Some are important prognostic or predictive factors for treatment (18). With the emergence and progress of molecular targeted therapies, it is appealing to assess systematically the genetic background of lung cancer patients. Comprehensive evaluation of mutations, however, is often hampered by the small sample size of bioptic material or availability of cytologic material only. Also, repeat biopsy at relapse to study potential resistant mutations (e.g. EGFR T790M mutation) is technically challenging and potentially dangerous. Detecting mutant tumor genes from peripheral blood circulation represents a potential alternative (114). Besides the detection of circulating tumor cells (115), a technique that still needs optimization, tumor DNA in the circulation is potentially a simpler and more attractive source of information (116).  
O
Techniques used nowadays for mutation detection, however, have several limitations. Traditional Sanger's method has relatively low sensitivity and specificity, especially when the tumor cells are contaminated with normal cells. Several real-time PCR based applications, such as scorpions-amplification refractory mutation system, are more sensitive (17, 18). The real-time PCR based applications, however, need designed primers or probes and can only detect prespecified known mutations. On the other hand, none of these techniques can quantify the presence of mutations, which can be important to evaluate the dynamics of tumor burden after treatment or evaluate gene copy number changes (e.g. MET).  
I
Invented initially for faster and more accurate genomic sequencing, next-generation sequencing (NGS) techniques, such as the Illumina genome analyzer, are applied to mutation discovery, DNA-protein interaction, mRNA expression, noncoding RNAs study, and metagenomics studies (113, 117). Through high-throughput sequence analysis and high coverage of depth, NGS may overcome the disadvantages mentioned above. NGS has been applied to sequence the cancer and leukemia genomes(118). NGS-based applications, RNA-seq and ChIP-seq, are superior to mRNA microarray and ChIP-on-chip technique, respectively, because they do not require prior knowledge of RNA and DNA sequences. It has been reported that NGS can detect EGFR mutations in malignant pleural effusion specimens with low tumor content and can measure the proportion of DNA molecules carrying the mutation as well (119). In summary, NGS could be an ideal platform to study genetic biomarkers of lung cancer in the circulation.  
D
In addition, urine samples are also ideal sources of biomarker researching. Urine samples have been evaluated for diagnosis of bladder cancer (120, 121). Giving that nucleic acids fragments can pass glomerulus and enter urine(122), it has been reported to be feasible to detect mutant genes from urine in patients with non-genitourinary origin solid tumors(123). Because microRNAs are potential important biomarkers of lung cancer(124, 125), are small in size (2226bp) to pass glomerulus, and are reported to be detectable in urine(126), it is interesting and reasonable to assess the role of urinary microRNA expression in lung cancer patients. 10.4.1 Blood sample analyses. If possible, blood will be collected in one EDTA tube (5ml purple top) and one Sodium Heparin Tube (10ml green top) upon enrollment in the trial. The samples will be stored at 4°C and sent to the following laboratory(s) for analyses: Dr. Udayan Guha V
Attn. Dr. Steven Lee National Cancer Institute Building 10, Room 8N258 10 Center Drive Bethesda, MD 20892 Phone: 301.402.0082 The blood will be centrifuged at 820g for 10 minutes. The plasma will be collected and stored in -80°C for further analyses that will include but are not limited to the following:
O
Circulating DNA mutation detection. The buffy coat, in which white blood cells and circulating tumor cells are located, will be isolated for possible circulating tumor cells analysis. DNA in the buffy coat will be extracted within 24 hours of blood sampling if no more analysis of circulating cells will be conducted. DNA will be extracted from 1 ml of plasma. A pilot study, which will enroll around 10 patients, will be conducted to evaluate the feasibility of detecting mutant genes in circulating tumor DNA. In the pilot study, mutant genes confirmed in tumor samples will be analyzed in the plasma by an Ilumina Genome Analyzer. If mutant genes are detected, the amount of mutant gene will be represented by the ratio of mutant gene over wild-type gene, adjusted by the copy number of a reference gene, such as LINE-1 or hTERT gene.  
I
After the pilot study and adjustment of experiments' conditions, circulating mutant genes will be measured regularly and their levels will be correlated with clinical treatment outcome. Screening of new mutations will be conducted if disease progression develops and its result will be compared with the result of tumor re-biopsy, if any. 10.4.2 Urine sample analyses. If possible, 5 ml of urine will be collected in a yellow top urine tube upon enrollment in the trial. The container will be stored at 4°C and sent to the following laboratory(s) for analyses: Dr. Udayan Guha Attn. Dr. Steven Lee National Cancer Institute Building 10, Room 8N258 10 Center Drive Bethesda, MD 20892 Phone: 301.402.0082  
D  
  
The urine will be used for further analyses that will include but are not limited to the following:
Urine micro RNA detection. Urine will be stored at 4°C and processed within 4 hours of collection. Urine will be mixed 1:1 with lysis buffer and frozen at -80°C for later DNA and RNA extraction. Urine RNA will be extracted. A panel of microRNA will be analyzed by realtime PCR, including let7a, mir21, mir29b, mir34a, mir34b, mir34c, and mir155. U6 snRNA will be measured as internal control. The association of microRNA results with patients' clinical characteristics and treatment outcomes will be examined. 10.4.3 Genomic Analysis V
Whole exome, transcriptome, and whole genome (only on a subset of patients) sequencing will be performed to identify somatic changes in tumor tissue if tissue is available after the other studies mentioned above are performed. Comparative genomic hybridization (CGH) analyses will be performed to interrogate the copy number gains and losses. DNA and RNA will be extracted from tumor samples and germ line DNA and RNA will be extracted from peripheral blood mononuclear cells from whole blood. DNA isolated from blood will be analyzed to identify the somatic and germline variants. Transcriptome sequencing will reveal expression at the RNA-level. The genomic analyses will be performed in ATC genome core facility, and in the laboratories of Drs. Meltzer, Raffeld and Guha. 10.4.3.1.1 DNA Sequencing O
Whole-exome multiregion spatial sequencing of DNA will be performed using a next-generation sequencing device and somatic mutations identified will be validated and verified using Sanger sequencing (NCI Frederick sequencing facility). Whole genome sequencing will be performed on a subset of samples based on the results of whole-exome and transcriptome sequencing. 10.4.3.1.2 RNA Sequencing I
Transcriptome profiling will be performed using RNA-Seq approach to study transcriptome complexity, and for identification of genes, structure of transcripts, alternative splicing, noncoding RNAs, and new transcription units (NCI Frederick sequencing facility). RNA-seq will also reveal expression of individual genes at the transcript-level. This will be correlated with quantitative protein expression by mass spectrometry.
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10.5 samples for genetic/genomic analysis

10.5.1 Description of how privacy and confidentiality of medical information/biological specimens will be maximized
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Tissue and blood samples will be coded. DNA, RNA and protein isolated from these tissues and cell lines and xenografts generated will all be similarly coded. Only personnel involved in this study will have access to both the code and the name of the patient. To help protect privacy, a Certificate of Confidentiality has been obtained from the National Institutes of Health. To facilitate genetic research, and for the purpose of publication of research work, data from genomic and proteomic studies may be deposited in appropriate public databases. Coded data will be deposited in a manner that the patient's identity cannot be traced.  
10.5.2 Management of Results Participants will be given the choice of whether or not they would like the Durable Power of Attorney (DPA) to receive notification if a gene variant known to cause or contribute to disease is identified. In cases where in the opinion of the principal investigator there is a finding of urgent importance to the health of the family members of the participants, the investigator will consult with an interdisciplinary group of geneticists, clinicians, and the NIH ethics board to determine whether the result needs to be divulged to immediate family members and the DPA will be notified by telephone or by mail. Attempts will be made to verify the results in a CLIA certified lab prior to notification. Results may become available for a period of up to two years. 10.5.3 Genetic Counseling V
Genetic counseling will be provided to the patient by genetic counseling services of the Clinical Center upon consent to the protocol and during one of the routine follow up visits to the clinical center. In the event a result of urgent importance to immediate family members has to be divulged, family members will be offered genetic counseling.
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11 data collection and evaluation

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The PI will be responsible for overseeing entry of data into in-house password protected electronic systems, C3D and LabMatrix, and ensuring data accuracy, consistency and timeliness. The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts. All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event.  
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Document AEs from the first study intervention through 30 days after the last study intervention. Beyond 30 days after the last intervention, only adverse events which are serious and related to the study intervention need to be recorded.  
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH
Intramural Records Retention Schecule as applicable.  
Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, this will be reported expeditiously per requirements in section **8.2.1**.
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11.2 genomic data sharing plan

Unlinked genomic data will be deposited in public genomic databases such as dbGaP in compliance with the NIH Genomic Data Sharing Policy.  
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12 study calendar

Baseline evaluations are to be conducted within 1 week prior to start of protocol therapy. Scans and x-rays must be done <4 weeks prior to the start of therapy. In the event that the patient's condition is deteriorating, laboratory evaluations should be repeated within 48 hours prior to initiation of the next cycle of therapy. The study calendar does not apply to individuals enrolled for *EGFR* germline mutation testing only; please refer to Section 5.6 for testing and follow-up of these individuals.  
| these individuals.  V Screening - Part 1 Molecular Profiling  and Correlative  Studies   | Screening - Part 2 Treatment Arm  Eligibility (see  Section 3.3)   | Treatment Cycles (see Section 5.3; clinic visits  may be extended to every 6 weeks for the  Erlotinib, AZD6244, and Lapatinib arms; every  8 weeks for the MK-2206 arm; and every 12  weeks for the Sunitinib arm) Off  treatment              |    |
|------------------------------------------------------------------------------------------|--------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----|
| Biopsy for                                                                               | X                                                                  | Upon disease progression per section 5                                                                                                                                                                                                         |    |
| molecular                                                                                | (optional)                                                         |                                                                                                                                                                                                                                                |    |
| profiling11 Targeted                                                                     | See section 5 for details regarding agent                          |                                                                                                                                                                                                                                                |    |
| therapies9                                                                               | administration and scheduling                                      |                                                                                                                                                                                                                                                |    |
| Informed  consent                                                                        | X                                                                  | N/A                                                                                                                                                                                                                                            |    |
| Demographics                                                                             | X                                                                  |                                                                                                                                                                                                                                                |    |
| NOS arm data10                                                                           | X                                                                  |                                                                                                                                                                                                                                                |    |
| Medical history9                                                                         | X                                                                  | Every clinic visit                                                                                                                                                                                                                             | X  |
| Concurrent  Meds9                                                                        | X                                                                  | Every clinic visit                                                                                                                                                                                                                             | X  |
| NIH Advance  Directives                                                                  | X                                                                  |                                                                                                                                                                                                                                                |    |
| Form14                                                                                   | O                                                                  |                                                                                                                                                                                                                                                |    |
| Physical exam9                                                                           | X                                                                  | Every clinic visit                                                                                                                                                                                                                             | X  |
| Vital signs9                                                                             | X                                                                  | Every clinic visit                                                                                                                                                                                                                             | X  |
| Height9                                                                                  | X                                                                  | N/A                                                                                                                                                                                                                                            |    |
| Weight9                                                                                  | X                                                                  | Every clinic visit                                                                                                                                                                                                                             | X  |
| Performance  status9                                                                     | X                                                                  | Every clinic visit                                                                                                                                                                                                                             | X  |
| CBC w/diff, plts9                                                                        | X                                                                  | Prior to each clinic visit                                                                                                                                                                                                                     | X  |
| Serum  chemistry1, 9                                                                     | X                                                                  | Prior to each clinic visit                                                                                                                                                                                                                     | X  |
| PT, aPTT9                                                                                | X                                                                  | I                                                                                                                                                                                                                                              | X  |
| Urinalysis9                                                                              | X                                                                  | X                                                                                                                                                                                                                                              |    |
| EKG9                                                                                     | X                                                                  | X                                                                                                                                                                                                                                              |    |
| BP3, 9                                                                                   | Weekly during cycle 1                                              |                                                                                                                                                                                                                                                |    |
| ECHO/MUGA4, 5, 9                                                                         | X                                                                  | Every other treatment cycle                                                                                                                                                                                                                    |    |
| Adverse event  evaluation9                                                               | X-----------------------------------------------------X            | X                                                                                                                                                                                                                                              |    |
| Tumor  measurements9                                                                     | X                                                                  | As clinically indicated DApproximately every 6, 8, or 12 weeks, as  defined for each treatment arm. See Section  5.8.2 for details. Documentation (radiologic)  must be provided for patients removed from  study arm for progressive disease. | X  |  
| Screening - Part 1 Molecular Profiling  and Correlative  Studies                                                                                                                      | Screening - Part 2 Treatment Arm  Eligibility (see  Section 3.3)   | Treatment Cycles (see Section 5.3; clinic visits  may be extended to every 6 weeks for the  Erlotinib, AZD6244, and Lapatinib arms; every  8 weeks for the MK-2206 arm; and every 12  weeks for the Sunitinib arm) Off  treatment   |    |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----|
| Radiologic                                                                                                                                                                            | Approximately every 6, 8, or 12 weeks, as                          |                                                                                                                                                                                                                                     |    |
| evaluation9                                                                                                                                                                           | X                                                                  | defined for each treatment arm. See Section                                                                                                                                                                                         | X  |
| 5.8.2 for details.                                                                                                                                                                    |                                                                    |                                                                                                                                                                                                                                     |    |
| V                                                                                                                                                                                     |                                                                    |                                                                                                                                                                                                                                     |    |
| B-HCG2, 9                                                                                                                                                                             | X                                                                  | As clinically indicated                                                                                                                                                                                                             |    |
| HgbA1c 6, 9                                                                                                                                                                           | X-------------------------------------------------------------X    |                                                                                                                                                                                                                                     |    |
| Correlative  studies7, 8, 9, 12                                                                                                                                                       | X                                                                  | Upon disease progression if repeat biopsy is performed                                                                                                                                                                              |    |
| EGFR germline  mutation                                                                                                                                                               | X                                                                  |                                                                                                                                                                                                                                     |    |
| testing13 1 :Sodium, potassium, chloride, CO2, BUN, creatinine, glucose, AST, ALT, alkaline phosphatase, bilirubin, albumin, total  protein, LDH, calcium, phosphorous and magnesium. |                                                                    |                                                                                                                                                                                                                                     |    |  
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1:Sodium, potassium, chloride, CO2, BUN, creatinine, glucose, AST, ALT, alkaline phosphatase, bilirubin, albumin, total protein, LDH, calcium, phosphorous and magnesium.  
2:Serum pregnancy test (women of childbearing potential). 3: Required only for patients enrolled on a Sunitinib treatment arm, per section 6.2.1.  
4: Required only for patients enrolled on a Lapatinib treatment arm, per section 6.1.5.  
5: Required for patients enrolled on a Sunitinib treatment arm who have known cardiac dysfunction at study entry and/or clinically observed adverse cardiac events.  
6: For patients enrolled on the MK-2206 arm, who require treatment of hyperglycemia per section 6.1.2, HgbA1c monitoring should be performed with each cycle of MK-2206 therapy 7: 1 5ml EDTA tube (purple top) and 1 urine sample collected in a (yellow top) urine tube. Page Betsy Morrow 10063 for pick up.  
8: 1 10ml Sodium Heparin Tube (green top). Call Zied Abdullaev for pick up at 301 451-2711. 9: N/A for patients assigned to the NOS arm.  
10: Required only for the patients assigned to the NOS arm.  
11: Including any other procedures necessary to do the biopsy, as clinically indicated.  
12: Correlative Studies: See Section 9 for details. 13: Selected patients only (see Section 5.6). Patient must meet eligibility requirements for *EGFR* germline mutation testing and have signed a separate consent form.  
14: As indicated in section **16.3**, all subjects ≥ age 18 will be offered the opportunity to complete an NIH advance directives form. This should be done preferably at baseline but can be done at any time during the study as long as the capacity to do so is retained. The completion of the form is strongly recommended, but is not required.  
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13 measurement of effect 13.1

For the purposes of this study, patients should be re-evaluated for response every 6 +/- 1 weeks. In addition to a baseline scan, confirmatory scans should also be obtained within 4 to 6 weeks following initial documentation of objective response.  
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Response and progression will be evaluated in this study using the new international criteria proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1) [Eur J Ca 45:228-247, 2009]. Changes in the largest diameter (unidimensional measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST criteria.
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13.1.1 definitions

Evaluable for toxicity. All patients will be evaluable for toxicity from the time of their first treatment.  
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Evaluable for objective response. Only those patients who have measurable disease present at baseline, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for response. These patients will have their response classified according to the definitions stated below. (Note: Patients who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.) Evaluable Non-Target Disease Response. Patients who have lesions present at baseline that are evaluable but do not meet the definitions of measurable disease, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for non-target disease. The response assessment is based on the presence, absence, or unequivocal progression of the lesions. 13.1.2 Disease Parameters I
Measurable disease. Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as >20 mm by chest x-ray, as >10 mm with CT scan, or >10 mm with calipers by clinical exam. All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). Note: Tumor lesions that are situated in a previously irradiated area will not be considered measurable.  
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Malignant lymph nodes. To be considered pathologically enlarged and measurable, a lymph node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed. Non-measurable disease. All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or pathological lymph nodes with ≥ 10 to <15 mm short axis), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial  
effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-measurable. Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts.  
'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same patient, these are preferred for selection as target lesions.  
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Target lesions. All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as target lesions and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease.  
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Non-target lesions. All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as non-target lesions and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up. 13.1.3 Methods for Evaluation of Measurable Disease I
All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam.  
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Clinical lesions. Clinical lesions will only be considered measurable when they are superficial
(e.g., skin nodules and palpable lymph nodes) and ≥10 mm diameter as assessed using calipers
(e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended. Chest x-ray. Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable. Conventional CT and MRI. This guideline has defined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm or less. If CT scans have slice thickness  
greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans). Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; however, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used should be optimized for the evaluation of the type and site of disease. Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body parts, and diseases. Ideally, the same type of scanner should be used and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be performed with breath-hold scanning techniques, if possible.  
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PET-CT. At present, the low dose or attenuation correction CT portion of a combined PET-CT is not always of optimal diagnostic CT quality for use with RECIST measurements. However, if the site can document that the CT performed as part of a PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be used for RECIST measurements and can be used interchangeably with conventional CT in accurately measuring cancer lesions over time. Note, however, that the PET portion of the CT introduces additional data which may bias an investigator if it is not routinely or serially performed.  
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Ultrasound. Ultrasound is not useful in assessment of lesion size and should not be used as a method of measurement. Ultrasound examinations cannot be reproduced in their entirety for independent review at a later date and, because they are operator dependent, it cannot be guaranteed that the same technique and measurements will be taken from one assessment to the next. If new lesions are identified by ultrasound in the course of the study, confirmation by CT or MRI is advised. If there is concern about radiation exposure at CT, MRI may be used instead of CT in selected instances.  
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Endoscopy, Laparoscopy. The utilization of these techniques for objective tumor evaluation is not advised. However, such techniques may be useful to confirm complete pathological response when biopsies are obtained or to determine relapse in trials where recurrence following complete response (CR) or surgical resection is an endpoint.  
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Tumor markers. Tumor markers alone cannot be used to assess response. If markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response. Specific guidelines for both CA-125 response (in recurrent ovarian cancer) and PSA response (in recurrent prostate cancer) have been published [JNCI 96:487-488, 2004; J Clin Oncol 17, 3461-3467, 1999; J Clin Oncol 26:1148-1159, 2008]. In addition, the Gynecologic Cancer Intergroup has developed CA-125 progression criteria which are to be integrated with objective tumor assessment for use in first-line trials in ovarian cancer [JNCI 92:1534-1535, 2000].  
Cytology, Histology. These techniques can be used to differentiate between partial responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as germ cell tumors, where known residual benign tumors can remain). The cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease.  
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FDG-PET. While FDG-PET response assessments need additional study, it is sometimes reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in assessment of progression (particularly possible 'new' disease). New lesions on the basis of FDG-PET imaging can be identified according to the following algorithm:  
- Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of PD
based on a new lesion.  
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- No FDG-PET at baseline and a positive FDG-PET at follow-up: If the positive FDGPET at follow-up corresponds to a new site of disease confirmed by CT, this is PD. If the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, additional follow-up CT scans are needed to determine if there is truly progression occurring at that site (if so, the date of PD will be the date of the initial abnormal FDGPET scan). If the positive FDG-PET at follow-up corresponds to a pre-existing site of disease on CT that is not progressing on the basis of the anatomic images, this is not PD.  
- FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy in cases where a residual radiographic abnormality is thought to represent fibrosis or scarring. However, it must be acknowledged that both approaches may lead to false positive CR due to limitations of FDG-PET and biopsy resolution/sensitivity. In addition, the FDG-PET may not be used to upgrade a response to a CR if the patient is actively being maintained on MK-2206, since the AKT inhibitor blocks intracellular glucose transport and may cause a negative FDG-PET result as an artifact, due to its pharmacodynamic target effect.  
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Note: A 'positive' FDG-PET scan lesion means one which is FDG avid with an uptake greater than twice that of the surrounding tissue on the attenuation corrected image. 13.1.4 Response Criteria 13.1.4.1 Evaluation of Target Lesions Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm.  
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Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum of diameters Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an  
absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study 13.1.4.2 Evaluation of Non-Target Lesions V
Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10 mm short axis) Note: If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response. Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase.  
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Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail in such circumstances, and the progression status should be confirmed at a later time by the review panel (or Principal Investigator). 13.1.4.3 Evaluation of Best Overall Response The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
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For Patients with Measurable Disease (i.e., Target Disease)  
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| Target                                                                                                                                                                                                         | Non-Target                                                                         | New     | Overall                                            | Best Overall Response           |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------|---------|----------------------------------------------------|---------------------------------|
| Lesions                                                                                                                                                                                                        | Lesions                                                                            | Lesions | Response                                           | when Confirmation is  Required* |
| CR                                                                                                                                                                                                             | CR                                                                                 | No      | CR                                                 | >4 wks. Confirmation**          |
| CR                                                                                                                                                                                                             | Non                                                                                    |         |                                                    |                                 |
| CR/Non-PD                                                                                                                                                                                                      | No                                                                                 | PR      | >4 wks. Confirmation**                             |                                 |
| CR                                                                                                                                                                                                             | Not                                                                                | No      | PR                                                 |                                 |
| evaluated                                                                                                                                                                                                      |                                                                                    |         |                                                    |                                 |
| V PR NonCR/NonPD/not  evaluated                                                                                                                                                                                                                | No                                                                                 | PR      |                                                    |                                 |
| SD                                                                                                                                                                                                             | Non                                                                                    |         |                                                    |                                 |
| CR/NonPD/not  evaluated                                                                                                                                                                                                                | No                                                                                 | SD      | documented at least  once >4 wks. from  baseline** |                                 |
| PD                                                                                                                                                                                                             | Any                                                                                | Yes or  | PD                                                 |                                 |
| No                                                                                                                                                                                                             | no prior SD, PR or CR                                                              |         |                                                    |                                 |
| O                                                                                                                                                                                                              |                                                                                    |         |                                                    |                                 |
| Any                                                                                                                                                                                                            | PD***                                                                              | Yes or  | PD                                                 |                                 |
| No                                                                                                                                                                                                             |                                                                                    |         |                                                    |                                 |
| Any                                                                                                                                                                                                            | Any                                                                                | Yes     | PD                                                 |                                 |
| *                                                                                                                                                                                                              | See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. |         |                                                    |                                 |
| **                                                                                                                                                                                                             | Only for non-randomized trials with response as primary endpoint.                  |         |                                                    |                                 |
| *** In exceptional circumstances, unequivocal progression in non-target lesions may  be accepted as disease progression. Note: Patients with a global deterioration of health status requiring discontinuation |                                                                                    |         |                                                    |                                 |  
  
  
For Patients with Non-Measurable Disease (i.e., Non-Target Disease)  
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| Non-Target Lesions                                                                                                                                      | New Lesions   | Overall Response   |    |
|---------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|--------------------|----|
| CR                                                                                                                                                      | No            | CR                 | D  |
| Non-CR/non-PD                                                                                                                                           | No            | Non-CR/non-PD*     |    |
| Not all evaluated                                                                                                                                       | No            | not evaluated      |    |
| Unequivocal PD                                                                                                                                          | Yes or No     | PD                 |    |
| Any                                                                                                                                                     | Yes           | PD                 |    |
| * 'Non-CR/non-PD' is preferred over 'stable disease' for non-target disease  since SD is increasingly used as an endpoint for assessment of efficacy in |               |                    |    |  
some trials so to assign this category when no lesions can be measured is not advised 13.1.5 Duration of Response V
Duration of overall response: The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented. Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements. 13.1.6 Progression-Free Survival O
PFS is defined as the duration of time from start of treatment to time of progression or death, whichever occurs first.
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14 data reporting / regulatory

Adverse event lists, guidelines, and instructions for adverse event reporting can be found in Section 7.2 (Adverse Events: List and Reporting Requirements).
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14.1 data reporting 14.1.1 method

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This study will be monitored by the Clinical Data Update System (CDUS) version 3.0. Cumulative CDUS data will be submitted quarterly to CTEP by electronic means. Reports are due January 31, April 30, July 31 and October 31. Instructions for submitting data using the CDUS can be found on the CTEP web site (http://ctep.cancer.gov/reporting/cdus.html). 14.1.2 Responsibility for Data Submission Study participants are responsible for submitting CDUS data and/or data forms to the Coordinating Center quarterly at least 2 weeks before CTEP's due date to allow time for Coordinating Center compilation, Principal Investigator review, and timely submission to CTEP
(see Section **14.1.1**). The Coordinating Center is responsible for compiling and submitting CDUS data to CTEP for all participants and for providing the data to the Principal Investigator for review.  
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14.2 ctep multicenter guidelines

This protocol will adhere to the policies and requirements of the CTEP Multicenter Guidelines. The specific responsibilities of the Principal Investigator and the Coordinating Center (Study Coordinator) and the procedures for auditing are presented in Appendix I.  
The Principal Investigator/Coordinating Center is responsible for distributing all IND Action Letters or Safety Reports received from CTEP to all participating institutions for submission to their individual IRBs for action as required. Except in very unusual circumstances, each participating institution will order DCTD-supplied 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 (PIO@ctep.nci.nih.gov) except for Group studies.
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14.3 collaborative agreements language

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The agent(s) supplied by CTEP, DCTD, NCI used in this protocol is/are provided to the NCI under a Collaborative Agreement (CRADA, Agent-CRADA, CTA, CSA) between the Pharmaceutical Company(ies) (hereinafter referred to as a "Collaborator(s)") and the NCI Division of Cancer Treatment and Diagnosis. Therefore, the following obligations/guidelines, in addition to the provisions in the "Intellectual Property Option to Collaborator" (http://ctep.cancer.gov/industryCollaborations2/default.htm) contained within the terms of award, apply to the use of the Agent(s) in this study:
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1. Agent(s) may not be used for any purpose outside the scope of this protocol, nor can Agent(s) be transferred or licensed to any party not participating in the clinical study. Collaborator(s) data for Agent(s) are confidential and proprietary to Collaborator(s) and shall be maintained as such by the investigators. The protocol documents for studies utilizing investigational Agents contain confidential information and should not be shared or distributed without the permission of the NCI. If a copy of this protocol is requested by a patient or patient's family member participating on the study, the individual should sign a confidentiality agreement. A suitable model agreement can be downloaded from: http://ctep.cancer.gov.  
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2. For a clinical protocol where there is an investigational Agent used in combination with (an)other investigational Agent(s), each the subject of different collaborative agreements, the access to and use of data by each Collaborator shall be as follows (data pertaining to such combination use shall hereinafter be referred to as "Multi-Party Data"):
a. NCI will provide all Collaborators with prior written notice regarding the existence and nature of any agreements governing their collaboration with NIH, the design of the proposed combination protocol, and the existence of any obligations that would tend to restrict NCI's participation in the proposed combination protocol.  
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b. Each Collaborator shall agree to permit use of the Multi-Party Data from the clinical trial by any other Collaborator solely to the extent necessary to allow said other Collaborator to develop, obtain regulatory approval or commercialize its own investigational Agent. c. Any Collaborator having the right to use the Multi-Party Data from these trials must agree in writing prior to the commencement of the trials that it will use the Multi-Party Data solely for development, regulatory approval, and commercialization of its own investigational Agent.  
3. Clinical Trial Data and Results and Raw Data developed under a Collaborative Agreement will be made available exclusively to Collaborator(s), the NCI, and the FDA, as appropriate and unless additional disclosure is required by law or court order. Additionally, all Clinical Data and Results and Raw Data will be collected, used, and disclosed consistent with all applicable federal statutes and regulations for the protection of human subjects including, if applicable, the Standards for Privacy of Individually Identifiable Health Information set forth in 45 C.F.R. Part 164.  
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4. When a Collaborator wishes to initiate a data request, the request should first be sent to the NCI, who will then notify the appropriate investigators (Group Chair for Cooperative Group studies, or PI for other studies) of Collaborator's wish to contact them. 5. Any data provided to Collaborator(s) for Phase 3 studies must be in accordance with the guidelines and policies of the responsible Data Monitoring Committee (DMC), if there is a DMC for this clinical trial.  
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6. Any manuscripts reporting the results of this clinical trial must be provided to CTEP by the Group office for Cooperative Group studies or by the principal investigator for non-Cooperative Group studies for immediate delivery to Collaborator(s) for advisory review and comment prior to submission for publication. Collaborator(s) will have 30 days from the date of receipt for review. Collaborator shall have the right to request that publication be delayed for up to an additional 30 days in order to ensure that Collaborator's confidential and proprietary data, in addition to Collaborator(s)'s intellectual property rights, are protected. Copies of abstracts must be provided to CTEP for forwarding to Collaborator(s) for courtesy review as soon as possible and preferably at least three (3) days prior to submission, but in any case, prior to presentation at the meeting or publication in the proceedings. Press releases and other media presentations must also be forwarded to CTEP prior to release. Copies of any manuscript, abstract and/or press release/ media presentation should be sent to:
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Jan M. Casadei, Ph.D., Chief, Regulatory Affairs Branch Cancer Therapy Evaluation Program Division of Cancer Treatment and Diagnosis, NCI 9609 Medical Center Drive Room 5-W532, MSC 9740 Bethesda, MD 20892-9740 [if US Postal Service] Rockville, MD 20850 [if non-USPS/private carrier] Tel. 240-276-6125 E-mail: casadeij@mail.nih.gov E-mail: ncicteppubs@mail.nih.gov  
  
The Regulatory Affairs Branch will then distribute them to Collaborator(s). No publication, manuscript or other form of public disclosure shall contain any of Collaborator's confidential/ proprietary information.
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15 statistical considerations 15.1 study

The primary objectives of this study are to determine the feasibility and efficacy of the use of molecular profile directed therapies in the treatment of advanced thoracic malignancies. The feasibility rate for the trial will be evaluated by determining the percentage of enrolled patients with a successful molecular profile determined. Efficacy will be determined by assessing if patients who have treatment assigned on the basis of their molecular profiling results will exhibit reasonable response rates to the drug selected for their particular profile.  
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Patients with NSCLC, SCLC, and thymic malignancies will be eligible for enrollment and each will be considered a separate cohort. In addition, on the basis of the molecular profiling results, one of 5 specific treatments could be assigned to each patient. The particular treatment assignments will be made according to a cascading schema as shown. If no profile of one of the 5 types listed is identified as being appropriate for a given patient, patients will be followed and treated in a separate 'other' (NOS) category.  
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With 3 disease types and 5 possible specific treatments for each, there are 15 possible treatment arms which will be under active consideration. For each of these 15, the study will be conducted as an optimal two-stage phase II trial (Simon R, Controlled Clinical Trials 10:1-10, 1989). In all but the arm for patients who have NSCLC and an EGFR mutation, the trial will be conducted in order to rule out an unacceptably low 10% clinical response rate (PR+CR; p0=0.10) in favor of a modestly high response rate of 40% (p1=0.40). For those 14 arms, with alpha=0.05 (probability of accepting a poor treatment=0.05) and beta = 0.10 (probability of rejecting a good treatment=0.10), the study will initially enroll 9 evaluable patients in each stratum and if 0 or 1 of the 9 have a clinical response, then no further patients will be accrued. If 2 or more of the first 9 have a response, then accrual would continue until a total of 20 patients have enrolled in that stratum. If there are 2-4 responses in 20 patients, this would be an uninterestingly low response rate in that stratum, while if there were 5 or more responses in 20 patients, then this would be sufficiently interesting to warrant further study in later trials. Under the null hypothesis (10% response rate), the probability of early termination is 77% for a given stratum.  
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In the arm for patients who have NSCLC and an EGFR mutation, the trial will be conducted in order to rule out an unacceptably low 30% clinical response rate (PR+CR; p0=0.30) in favor of a modestly high response rate of 60% (p1=0.60). The target is higher for these patients since a higher response rate is known to be possible for these patients. For that one arm, with alpha=0.05 (probability of accepting a poor treatment=0.05) and beta = 0.10 (probability of rejecting a good treatment=0.10), the study will initially enroll 10 evaluable patients in each stratum and if 0 to 3 of the 10 have a clinical response, then no further patients will be accrued. If 4 or more of the first 10 have a response, then accrual would continue until a total of 28 patients have enrolled in that stratum. Under the null hypothesis (30% response rate), the probability of early termination is 65% for patients in this stratum.  
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As feasibility is an important endpoint as well, the trial will include an early stopping rule for accrual to specific arms which are not enrolling adequate numbers of patients. After 18 months from the date the first patient enrolls on the trial in any arm, if there are arms with no more than a  
single patient, then further accrual to that arm will end. This will prevent continued accrual to arms which are very unlikely to be able to treat sufficient patients for evaluating the first stage (e.g., 9 or 10 patients) within 5 years.
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15.2 sample size/accrual rate

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With a maximum of 20 patients per stratum, up to 300 evaluable patients may be treated in specific arms. All patients in the 'other' (NOS) stratum will be evaluated and have their data analyzed by disease and type of treatment to the extent possible. As they are considered entirely exploratory, no specific targets for accrual will be used for the patients in the 'other' (NOS) stratum. For the secondary objective of determining the frequency of *EGFR* germline mutations in families with high susceptibility to lung cancer, we plan to enroll 15 families over a period of a year; however, specific accrual targets are not provided due to the exploratory nature of this endpoint. In order to enroll up to 300 patients in the specific arms on the basis of molecular profiling results, it is anticipated that based on the rates of mutations that occur in the genes used for treatment assignments, approximately 600 patients may need to be enrolled onto the trial. It is anticipated that up to 10 patients per month may be enrolled onto this trial from all participating sites and in all diseases combined, and thus approximately five years may be required to enroll up to 600 patients. Far fewer patients may be enrolled if the treatments fail to allow accrual to the second stage of the trial or if some arms close to accrual due to feasibility issues.  
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15.3 stratification factors n/a 15.4

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In order to accomplish the secondary objective of identifying molecular profiles in patients with NSCLC, SCLC or thymic malignancies and characterize their natural histories, clinical course and response to treatment, we will follow all patients that undergo molecular profiling from the time that they are enrolled in the trial until the time of death. Demographic information such as age, gender and race will be tabulated. The frequencies of oncogenic mutations will be assessed. The exact binomial two-sided 95% confidence interval for each mutation rate will be reported. Kaplan-Meier curves for progression free survival, duration of response, and overall survival will be calculated from the patients treated in the different treatment arms of this study (erlotinib, lapatinib, sunitinib, AZD6244, MK2206, or NOS). The results of this analysis may be compared in an informal manner to any similarly defined curves available from other published studies in comparable patients with the same disease. Expression levels of various proteins from pre- and post-treatment samples will be compared using an appropriate nonparametric test and pre-post differences between responders and non-responders using a Wilcoxon rank sum test. These latter evaluations will be considered exploratory and the resulting p-values will be presented as being exploratory and without adjustment for multiple comparisons.  
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For the secondary objective of determining the frequency of *EGFR* germline mutations in families with high susceptibility to lung cancer, the analyses will be performed in an exploratory manner. Hence, statistical power calculations have not been provided.
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15.5 reporting and exclusions 15.5.1

All patients will be evaluable for toxicity from the time of the biopsy for molecular profiling is performed. Patients who are subsequently enrolled in the NOS arm will not be evaluable for toxicity. 15.5.2 Evaluation of response V
All patients included in one of the experimental arms of the study must be assessed for response to treatment, even if there are major protocol treatment deviations. Each patient will be assigned one of the following categories: 1) complete response, 2) partial response, 3) stable disease, 4) progressive disease, 5) early death from malignant disease, 6) early death from toxicity, 7) early death because of other cause, or 9) unknown (not assessable, insufficient data). [Note: By arbitrary convention, category 9 usually designates the "unknown" status of any type of data in a clinical database.]
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All of the patients who met the eligibility criteria for a particular treatment arm (with the exception of those who received no study medication) should be included in the main analysis of the response rate. Patients in response categories 5-9 should be considered to have a treatment failure (disease progression). Thus, an incorrect treatment schedule or drug administration does not result in exclusion from the analysis of the response rate. Precise definitions for categories 5-9 will be protocol specific. All conclusions should be based on all eligible patients. Subanalyses may then be performed on the basis of a subset of patients, excluding those for whom major protocol deviations have been identified (e.g., early death due to other reasons, early discontinuation of treatment, major protocol deviations, etc.). However, these subanalyses may not serve as the basis for drawing conclusions concerning treatment efficacy, and the reasons for excluding patients from the analysis should be clearly reported. The 95% confidence intervals should also be provided.  
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16 human subjects protections 16.1

This study will be open to all individuals with advanced thoracic malignancies (lung cancer and thymic malignancies) regardless of gender, ethnicity, or race provided that the aforementioned inclusion and exclusion criteria are met. For safety reasons, only pregnant women and children are excluded from this study. This study will be recruited through internal referral, our local physician referral base, and through various cancer information hotlines (i.e., Clinical Studies Support Center, 1-800-4Cancer). This is a trial designed to evaluate the efficacy of different targeted therapies in patients with advanced thoracic malignancies. Patients should realize that we are hopeful that they may gain benefit from this study, but there is no objective evidence to support our optimism at this time. Subjects from both genders and all racial /ethnic groups are eligible for this study if they meet the eligibility criteria outlined in Section 3. To date, there is no information that suggests that differences in drug metabolism or disease response would be expected in one ethnic group compared to another. Efforts will be made to extend accrual to each representative population, but in this preliminary study, a balance must be struck between D  
patient safety considerations and limitations on the number of individuals exposed to potentially toxic and/or ineffective treatments on the one hand and the need to explore racial/ethnic aspects of clinical research on the other hand. If differences in outcome that correlate to ethnic identity are noted, accrual may be expanded or a follow-up study may be written to investigate those differences more fully.
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16.2 justification for exclusions

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Due to lack of knowledge of the effects of most or all of the targeted agents included in this trial on the fetus or on infants, as well as the possibility of teratogenic effects, pregnant and nursing women will be excluded from this trial. Patients with unstable or serious medical conditions (ongoing or active infection, symptomatic congestive heart failure (AHA Class II or worse), unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements) are excluded due to the possibility that the agents used may worsen their condition and the likelihood that the underlying condition may obscure the attribution of adverse events with respect to protocol therapy. 16.2.1 Participation of Children O
Patients under the age of 18 will be excluded from study due to the low occurrence of these oncologic histologies in the pediatric population. In addition, the risk of exposure to an investigational agent without proven benefit in the targeted histologies supports excluding children until additional safety and efficacy data is available. Patients under the age of 18 will be excluded from germline mutation testing because of the infrequency of lung cancer in this age group and because the results would not influence the medical management of individuals under the age of 18.
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16.3 participation of subjects unable

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Adults unable to give consent are excluded from enrolling in the protocol. However, re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (section **16.4**), all subjects ≥ age 18 will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent Assessment Team (ACAT) for evaluation as needed for the following: an independent assessment of whether an individual has the capacity to provide consent; assistance in identifying and assessing an appropriate surrogate when indicated; and/or an assessment of the capacity to appoint a surrogate. For those subjects that become incapacitated and do not have pre-determined substitute decision maker, the procedures described in NIH HRPP SOP 14E for appointing a surrogate decision maker for adult subjects who are (a) decisionally impaired, and (b) who do not have a legal guardian or durable power of attorney, will be followed.  
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16.4 evaluation of benefits and

The potential benefit to a patient who enters study is a reduction in the bulk of his/her tumor, which may or may not have a favorable impact on symptoms and/or survival. Potential risks  
include the possible occurrence of any of a range of side effects that are listed in the pharmaceutical section and the consent document. The procedure for protecting against or minimizing risks will be to medically evaluate patients on a regular basis as described earlier. As of 3/22/2016, the treatment arms of this study have been closed. The study remains open under NIH Intramural IRB for the molecular profiling arm only. Participating in the molecular profiling arm is associated with risks that may accompany the biopsy required for genetic testing. These risks include bleeding or infection at the biopsy site as well as potential radiation exposure if the biopsy is done with CT guidance. Additionally, participation comes with the risk of discovering new genetic results that may have implications on the participant and/or family members. The benefits of participation include gaining additional genetic knowledge which may provide guidance in cancer treatment options provided outside of this study. Pre and post testing genetic counseling will be provided as these results are disclosed to provide assistance in the handling of these results.  
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16.5 risk/benefit analysis

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As of 3/22/2016, the treatment arms of this study have been closed, but study remains open under NIH Intramural IRB for molecular profiling arm only. For adults in the molecular profiling arm, including those who become unable to consent, there is more than minimial risk in participation associated with the biopsy with the possibility of direct benefit by way of return of genetic findings that may provide additional treatment options outside of this study.
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16.6 consent and assent process

An associate or principal investigator on the trial will inform patients of the purpose, alternatives, treatment plan, research objectives and follow-up of this trial. The patient will be provided an IRB-approved consent for review and signature and his/her questions will be answered. After a decision is made to enroll into the study, a signature will be obtained from the patient at a subsequent visit. The original of the signed informed consent will be placed in the patient's medical record and a copy will be held in the research record.  
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All patients must have a signed informed consent form and an on-study (confirmation of eligibility) form filled out and signed by a participating investigator before entering on study. The proband as well as all family members undergoing germline genetic testing will sign a separate informed consent for germline mutation testing. For deceased family members of the proband with history of lung cancer, who have available tissue, consent will be obtained from the next of kin. 16.6.1 Telephone Consent (for amendments only):
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The informed consent document will be sent to the subject. An explanation of the study will be provided over the telephone after the subject has had the opportunity to read the consent form. The subject will sign and date the informed consent. The original informed consent document will be sent back to the consenting investigator who will sign and date the consent form with the date the consent was obtained via telephone. A fully executed copy will be returned via mail for the subject's records.  
The informed consent process will be documented in the medical record.
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16.7 data and safety monitoring

Any new significant finding that may affect the patient's willingness to continue in the study will be shared with patients. Data will be monitored regularly by the principal investigator in order to identify significant toxicity trends. Confidentiality will be maintained as much as possible, consistent with applicable regulations. Names of participants or identifying material will not be released without patient permission, except when such release is required by law. No patient's name or identifying information will be released in any publication or presentation. Records are maintained according to current legal requirements and are made available for review according to the requirements of the Food and Drug Administration (FDA) or other authorized user, only under guidelines established by the Federal Privacy Act.  
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This study will also be monitored by the National Cancer Institute Safety Monitoring Committee (SMC). The Coordinating Center Principal Investigator will obtain the required data from all participating sites and will report to the SMC annually, or as otherwise directed by the SMC.  
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17 references

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untitled h1
18 appendices appendix a study

This appendix contains background information about the study agents. This was provided by CTEP as a summary of relevant data for each study agent. More detailed information about each agent can be found in their respective investigational brochure.  
18.1.1 CUP supplied agents  18.1.1.1 Mk-2206
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The PI3K/AKT pathway is downstream of the common growth factor tyrosine kinase receptors (RTK), including EGFR, HER2, IGFR, etc., and is a likely driver of tumor progression in most carcinomas (127-129). AKT protein kinase is activated in a substantial proportion of human solid tumors (breast, endometrial, ovarian, prostate, pancreatic, colon, gastric, and non-small cell lung cancer [NSCLC]). Upregulation of AKT can be caused by direct amplification/mutation of AKT, or by overexpression of RTKs, PI3K and RAS, and/or by inactivation of the tumor suppressor PTEN. Because of its key function in cell survival, AKT plays a pivotal role in rendering tumor cells insensitive or resistant to chemotherapy or targeted agents.  
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The rationale for the use of an AKT inhibitor in treatment of various malignancies is included in the following references (3, 127-159). MK-2206 is the first allosteric inhibitor of AKT to enter clinical development (3). MK-2206 demonstrated AKT inhibition and antiproliferative activity as single agent and in combination with other agents in multiple human cancer cell lines, such as breast, ovarian, lung, and prostate. MK-2006 synergized antitumor effects of docetaxel, erlotinib, and carboplatin in vivo in various human tumor xenograft models. Mechanism of Action I
MK-2206 is a selective allosteric inhibitor of AKT (3). MK-2206 does not bind to the active site of AKT, and consequently does not compete with either ATP or peptide substrate for binding to AKT. It is equally potent against the two human AKT isoforms, AKT1 and AKT2, and ~5-fold less potent against AKT3.  
Nonclinical Studies In Vitro Activity Studies In vitro single-agent activity of MK-2206 D
In an in vitro kinase assay with GSK3 alpha peptide as substrate, MK-2206 strongly inhibited kinase activity of the three human isoforms of AKT, AKT1, 2, and 3, with 50% inhibitory concentration (IC50) values of 8, 12, and 65 nM, respectively. MK-2206 exhibited no inhibition (IC50>50 micromol/L (mcM) against the (pleckstrin-homology domain)-deletion mutants of AKT, indicating that this domain is essential for binding MK-2206 to AKT. Apart from AKT, MK-2206 was tested at a single concentration of 1 mcM against a panel of ~250 proteins including kinases without demonstrating significant (≥50%) inhibition against any kinase. The antiproliferative potency of MK-2206 was evaluated against a panel of tumor cell lines using in vitro proliferation and viability assays. Among 52 cell lines, 18 cell lines were highly  
sensitive (IC50<1 mcM), 7 were moderately sensitive (IC50=1-5 mcM), and 27 cell lines were insensitive (IC50>5 mcM) to MK-2206. Highly sensitive cell lines included breast, ovarian, prostate, NSCLC, small cell lung cancer (SCLC), gastric, and endometrial cancer. At least one of the following genetic defects was represented in the majority of sensitive cell lines: PTEN mutation, PI3KCA mutation, AKT amplification, or genomic amplification of HER2 or MET. Nine of 27 cell lines that were insensitive to MK-2206 had either PTEN or PI3KCA mutation, or were deficient in PTEN protein. Among eight cell lines carrying RAS or BRAF activating mutation, six were insensitive to MK-2206. MK-2206 activity in combination with other agents V
In a proliferation/viability assay, various degrees of synergism between MK-2206 and lapatinib (a dual EGFR/HER2 inhibitor) were observed in eight breast cancer cell lines. MK-2206 and docetaxel demonstrated additive to synergistic antiproliferative activity against nine breastcancer cell lines, however, the effects were dependent on agent sequence. Enhancement of antitumor activity occurred only when cells were treated first with docetaxel and then exposed to MK-2206. In contrast, co-administration of the two agents resulted in antagonism. Combination of MK-2206 with erlotinib produced various degrees of synergism in nine NSCLC cell lines, including A431 epidermoid cells overexpressing EGFR.  
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MK-2206 synergized antitumor activity of carboplatin, gemcitabine, doxorubicin, camptothecin, or 5-FU in ovarian, prostate and NSCLC cell lines. Antitumor activity of MK-2206/carboplatin, demonstrated in the ovarian cell line A2780, was accompanied with enhanced apoptosis (cleavage of caspases 3 and 7). However, the effect was dependent on the sequence of agent administration, as it occurred only when cells were exposed to carboplatin before or simultaneously with MK-2206. Enhancement of apoptosis did not occur when MK-2206 preceded carboplatin.  
In Vivo Activity Studies I
MK-2206 monotherapy administered as a single dose (10-240 mg/kg) to mice bearing human ovarian tumor (A2780) potently inhibited phosphorylation of AKT1/2 in blood as well as in tumor tissue. After a single dose of 30, 120, or 240 mg/kg of MK-2206, ≥80% inhibition of AKT1/2 was achieved in peripheral blood; inhibition persisted at this level for 6 and 24 hours at the 120- and 240-mg/kg dose, respectively. Pronounced inhibition (70-90%) of phosphorylation of AKT1/2 (pAKT1/2), lasting for at least 24 hours, was detected also in tumor tissue following the single 120- or 240- mg/kg dose. MK-2206 combinations with docetaxel, carboplatin, or erlotinib exhibited significantly more potent antitumor activity than each agent in monotherapy settings. For example, a combination of MK-2206 and docetaxel, administered for 4 weeks as one weekly intravenous (IV) dose of docetaxel (30 mg/kg) followed 24 hours later by MK-2206 (240 or 480 mg/kg) given orally (PO) once a week (QW), produced strong antitumor responses in the A2780 ovarian-cancer xenograft mouse model. MK-2206 monotherapy was ineffective at all doses. Although the combination was significantly more effective than docetaxel monotherapy, responses did not endure beyond the treatment period. Tumor regression was also observed in PC-3 prostate xenograft model following sequential treatment with docetaxel and MK-2206 (24 hours later). MK-2206 (120 mg/kg) was given three times per week (days 1, 3, 5) and docetaxel (5 mg/kg) once weekly on day 0. Inhibition of tumor growth was also enhanced D  
when MK-2206 was combined with carboplatin and erlotinib in the NSCLC H460 and H292 xenograft models, respectively. Overall, the agents' combinations were well-tolerated at the effective dose levels, although transient 10-20%-weight loss was observed in some animals.  
Pharmacokinetics V  
| Cl (mL/min/k g)   |
|-------------------|  
Pharmacokinetic (PK) parameters of MK-2206 in the rat, dog, and monkey are summarized in Table 18. Across the species, MK-2206 showed moderate plasma clearance (Cl) and high volume of distribution at steady state (Vdss) and elimination half-life (t1/2) ranging from 4 hours in rats to >12 hours in dogs and monkeys.  
| Vdss  (L/kg )   |
|-----------------|  
Species Dose  
(mpk)  
Cl  
O  
Rhesus 0.5 10.8 11 14 IV Route PO Route Rat 2 27.6 9 4.2 10 2.1 0.29 5 20 Dog 0.5 7.7 8 12.  
5  
2 8.89 0.38 4 83  
| t1/2     | Dose   | AUC          |            |    |
|----------|--------|--------------|------------|----|
| (h)      | (mpk)  | Cmax  (mcM ) | tma x  (h) | F  |
| (mcM∙h ) | (%)    |              |            |    |
| 100      | 35.2   | 2.76         | 2.7        | 35 |
| 2        | 8.89   | 0.38         | 4          | 83 |
| 5        |        |              |            |    |  
I  
  
  
  
Cl: clearance; Vdss: volume of distribution at steady state; t1/2: elimination half-life; AUC: the area under the time-concentration curve; Cmax: maximum drug concentration in plasma; tmax: time needed to achieve Cmax; F: oral availability; mcM: micromol/L; mpk: mg per kg; Vehicle: intravenous (IV): DMSO, oral route (PO): 0.5% methylcellulose (10 mpk),  
   
MK-2206 was significantly bound to plasma proteins (ranged from 96% to 88% in mice>rats>dogs>monkeys>humans) with moderate Cl in rats, but relatively low Cl in dogs and monkeys.  
D  
  
The oral availability of MK-2206 was acceptable in rats (20%-35%) and better in dogs (83%). Plasma Cl in rats occurred primarily by direct glucuronidation with <3% of MK-2206 excreted in feces. In dogs, elimination occurred via multiple metabolic pathways including oxidation followed by glucuronidation, direct glucuronidation, and formation of a carbamoyl glucuronide. Relative to rats, a higher fraction (30%) of parent compound MK-2206 was excreted (urine, bile, and feces) in dogs. Significant intestinal secretion was observed in dogs. No metabolites of MK-2206 with biologic activity were noted.  
MK-2206 was also metabolized by oxidation in human liver microsomes, primarily via the 3A4 isoenzyme of the P450 cytochrome (CYP) enzyme complex. MK-2206 was neither a potent inhibitor nor inducer of human CYPs. Transport experiments in the P-glycoprotein (P-gp)- transfected cell lines (L-MDR1) suggested that MK-2206 could be a P-gp substrate. In addition, MK-2206 demonstrated weak inhibition of vectorial transport of digoxin in L-MDR1 cells (IC50 of 13.4 mcM).  
V
Toxicology In the 10-day tolerability studies, dose-limiting toxicities (DLTs) were observed at the dose of ≥200 mg/kg/day and ≥15 mg/kg/day in rats and dogs, respectively. In a 4-week safety study in dogs, MK-2206 was administered at 2.5, 5, or 10 mg/kg PO every-other-day (QOD) followed by a 2-week recovery period. The 10-mg/kg dose was poorly tolerated as manifested by severe body-weight loss and other physical and histomorphological signs of toxicity, which required cessation of dosing by the end of the second week of treatment. The 5-mg/kg dose was tolerated and although treatment-related toxicities occurred, they were transient. As no significant toxicities were observed in dogs at the 2.5-mg/kg dose, this dose was defined as NOAEL (no observed adverse effect level). Exposure at this level characterized by the maximum plasma concentration (Cmax) and the area under the time-concentration curve (AUC0-48h) corresponded to 0.37 mcM and 8.52 mcM*hr, respectively. A potential safety concern associated with MK-2206 therapy is prolongation of the QT-corrected (QTc) interval, which seemed to be dose-dependent. While QTc-prolongation was persistent in dogs at the 10-mg/kg dose, it declined and returned to baseline between 24 to 72 hours post-dosing at 5 and 2.5 mg/kg. No cardiovascular changes were observed at the 1-mg/kg dose of MK-2206 (Cmax of 0.092 mcM and AUC0-24h of 1.6 mcM) within 48 hours following dosing in dogs.  
O
I
A tissue distribution study demonstrated that [14C]MK-2206 was widely distributed in Sprague Dawley (albino) and Long Evans (pigmented) rats except for the central nervous system. The majority of the radioactivity went into the muscle, liver and skin shortly after dosing. In Sprague Dawley and Long Evans rats, the radioactivity in most tissues was comparable. It declined in parallel to that in blood and became negligible 3 days post-dose. However, the radioactivity was more sustained at higher levels in the skin and the uveal tract of the eye in Long Evans than in Sprague Dawley rats. The concentration (ng equivalent [14C]MK-2206/g tissue) in the skin and the uveal tract of Long Evans rats was 37- and 84-fold higher than that in the respective tissues of Sprague Dawley rats 24 hours post-dose.  
D
An additional safety concern involves MK-2206-induced hyperglycemia and hyperinsulinemia. MK-2206 induced hyperglycemia in all preclinical species tested. In the most sensitive species, the dog, the glucose level was elevated by 24%-35%, when MK-2206 was administered at 5 mg/kg QOD for 4 weeks (AUC0-48 of 19.6 mcM∙h and Cmax of 0.73 mcM). Such exposure is expected to correspond with human exposures achievable at the upper MK-2206 dosing range. Results from genetic toxicology assays demonstrated that MK-2206 was neither genotoxic nor mutagenic. Clinical Development of MK-2206  
Preliminary clinical PK/pharmacodynamic and safety experience is derived from a Merck (Merck Sharp and Dohme)-sponsored phase 1 study in healthy volunteers (HVs) and company phase 1 studies in patients with advanced solid tumors. Projections derived from preclinical PK and metabolism studies in dogs suggested that the target exposure corresponding to a plasma concentration of ≥100 nM MK-2206 over 8 hours and AUC0-48h of ~2 mcM∙h in humans could be attained by MK-2206 dosed at 30-70 mg QOD on a 28-day cycle schedule. Clinical PK/pharmacodynamic data confirmed that the MK-2206 dose of 60 mg QOD conferred substantial and lasting inhibition of AKT as measured in tumor biopsies from cancer patients. The 60-mg QOD dose level is being currently investigated as the maximum tolerated dose (MTD) in the expanded cohort of patients.  
V
Preclinical and clinical experience from Merck-sponsored studies suggests potential benefit of a less frequent dosing schedule (i.e., QW). It is potentially feasible to administer MK-2206 at higher dose levels on a less frequent dosing schedule to maximize significant or peak target inhibition. This approach may also alleviate DLTs (e.g., skin rash) associated with accumulated exposure to MK-2206. Dose escalation on a QW schedule is currently being evaluated at the MK-2206 doses ranging from 90-300 mg. Clinical Pharmacokinetics O
The clinical PK for MK-2206 was evaluated in HVs receiving a single dose (0.25-100 mg) and in cancer patients given either a 30-, 45-, 60-, 75-, or 90-mg dose on the QOD schedule or 90-, 135-, 200-, 250-, or 300-mg dose on the QW schedule. Although Day 1 AUC0-48h and Cmax values achieved at ≤90 mg in cancer patients overlapped with the ranges observed in HVs, overall, MK-2206 exposures in cancer patients trended on average somewhat higher than those observed in HVs. In all cohorts evaluated at ≤200 mg QW, exposures after the first and last dose in Cycle 1 were below the dog NOAEL AUC0-48h and Cmax values of 8.52 mcM*hr and 365 nM, respectively. The mean first dose MK-2206 AUC0-48h and Cmax were 1.77 mcM*hr and 62.2 nM, respectively, for 60 mg QOD, and 14.8 mcM*hr and 466 nM, respectively, for 300 mg QW. The dog NOAEL exposures were exceeded in humans following the first dose of 300 mg QW.  
I
The variability in AUC0-48h and Cmax following the first dose, where it could be assessed, was low to moderate across all dose levels, with the coefficient of variation (CV) values ranging from approximately 10%–60%. From a visual inspection of the data there does not appear to be a substantial or consistent departure from dose proportionality for either AUC0-48hr or Cmax following the first dose up to the 300-mg dose level.Tmax and apparent terminal t1/2 values from cancer patients were generally within the ranges observed in HVs. Median Tmax values ranged from 4–10 hours across all dose regimens, and harmonic mean apparent terminal half-life (t1/2) values ranged from approximately 60–80 hours, with the exception of the 90 mg QOD cohort. At 90 mg QOD, apparent terminal t1/2 was assessable in one patient and was approximately 50 hours.  
D
Clinical Efficacy/Pharmacodynamics Preliminary pharmacodynamic results in cancer patients indicate that phosphorylation of AKT in whole blood is substantially inhibited at all dose levels evaluated on the QOD and QW schedule.  
Additionally, preliminary results indicate that substantial pAKT inhibition was demonstrated in tumor tissue at 60 mg QOD. As there is a causal relationship between the development of hyperglycemia/hyperinsulinemia and mechanism of AKT inhibition, such events could potentially implicate pharmacodynamic activity of MK-2206. In cancer patients, reversible grade 1/2 hyperglycemia was observed across all dose levels in a total of 59 patients. Hyperinsulinemia occurred in 26 patients who received MK-2206 60 mg QOD. From a preliminary analysis, these adverse events (AEs) do not appear to be dose-dependent. Neither hyperglycemia nor hyperinsulinemia was observed at any single dose in HVs.  
V
Thus far, no formal efficacy studies have been performed with MK-2206; however, in patients with advanced solid tumors, early indications of antitumor activity included substantial decreases in CA125 in some patients with ovarian cancer and PSA stabilization in some prostate cancer patients. Minor RECIST responses, e.g., <30% decreases in tumor size, have also been observed in a patient with melanoma (16%), a patient with pancreatic cancer (23%), and in a patient with neuroendocrine tumor (20%). No partial responses, e.g., confirmed >30% decreases in tumor size, have been observed. Clinical Toxicology O
Preclinical efficacy and safety studies and preliminary safety data from the clinical studies support the use of MK-2206 via the oral route, both as monotherapy and in combination with other anticancer agents. Overall, MK-2206 has been generally well-tolerated when administered as a single PO dose (0.25-100 mg) to HVs, or to cancer patients as the 30-60 mg PO dose on the QOD schedule and the 90-200 mg PO dose on the QW schedule. Mild to moderate skin rash was observed in 21 of 42 patients (50.0%) and severe skin rash was observed in 5 of 42 patients (11.9%) at the dose of 60 mg QOD. Skin rash resolved following the 1- to 2- week therapy break. The higher doses evaluated in oncology patients (i.e., 75 and 90 mg QOD and 300 mg QW) were not tolerated and resulted in DLT of grade 3/grade 4 skin rash.  
I
Mild to moderate mucositis and conjunctivitis were associated with rash. The supportive-care measures included hydration, topical steroid preparations, oral corticosteroids, oral antihistamines, and oral antibiotics. Other common AEs included nausea, fatigue, vomiting, and diarrhea. These AEs were mild to moderate and in most cases were resolved by the standard supportive care and did not require therapy modifications. Hyperglycemia and hyperinsulinemia, both expected mechanism-based AEs, were observed in approximately 76% and 57% of patients, respectively, receiving MK-2206 on the QOD schedule. Episodes were generally mild, transient, and did not require therapeutic intervention. Importantly, administration of insulin may not counteract MK-2206-induced hyperglycemia due to mechanism-based insulin resistance. In this case, hydration and oral antihyperglycemic agents can be used as the supportive-care measures. Grade 3 hyperglycemia occurred in one patient who received 60 mg QOD and required treatment with PO antihyperglycemic medication. Grade 4 hyperglycemia was reported in one patient who received MK-2206 45 mg QOD in combination with erlotinib. In addition to supportive care measures, blood glucose management included administration of insulin and PO antihyperglycemic medication.  
D  
Grade 1/grade 2 prolongation of QTc-interval was observed in 14 out of 64 patients (21.9%) with available 12-lead ECG data. Prolongations ≥30 msec but <60 msec occurred in 4 of these patients. Grade 3 QTc prolongation was reported in one patient who received 135 mg QW. Episodes of QTc interval prolongation were in general isolated with no apparent dependency on dose or exposure levels, and were not considered clinically significant by investigators and were not reported as adverse experiences. Eleven patients experienced sinus bradycardia (<50 bpm) during Holter or ECG monitoring. These events were asymptomatic and were not clinically significant. While a causal relationship between these events and administration of MK-2206 is uncertain, similar side effects were seen preclinically in conscious dogs. Consequently, patients with a history or current evidence of heart disease should be excluded from enrollment on MK2206 trials. Standard 12-lead ECG measurements should be performed at the protocol-specified time-points.  
V
Developmental/Reproductive Toxicity Developmental and reproductive toxicity studies of MK-2206 have not been performed thus far. MK-2206 was not tested in pregnant or breast-feeding women. Women of child-bearing potential and men participating in clinical studies of MK-2206 must use appropriate contraception, including abstinence and double-barrier methods, throughout MK-2206 therapy. In preclinical mutagenicity studies, MK-2206 was neither genotoxic or mutagenic.  
O
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drug interactions

No clinical drug interaction studies have been performed with MK-2206. Oxidative metabolism of MK-2206 in human liver microsomes was catalyzed primarily by CYP3A4. MK-2206 was not an inhibitor of the major CYPs. MK-2206 was shown to be a P-gp substrate.
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weekly dosing

I
Preclinical studies in animal models have shown evidence of improved efficacy of the once weekly (QW) dosing in mono-therapy using the A2780 xenograft (PTEN mutant) model in two independent studies. Additionally, QW dosing of MK-2206 results in similar or enhanced single agent and combination efficacy in NCI-H-292 Xenografts. In the phase I clinical trial PN002, MK-2206 showed a Cmax = 170 nM (after 1st dose) and Cmax = 225 nM (SS), which could potentially lead to better tumor penetration, more tumor cytotoxicity and better efficacy in comparison to the every other day (QOD) mode of administration. Mean Coverage above target PK was 133 hours (out of 168 hours), which may potentially alleviate skin toxicity. Furthermore, this dosing schedule is significantly more convenient for the patient. More detailed information and updates about the rationale for the use of QW dosing can be found in the investigational brochure.  
D
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16.1.1.2 azd6244

AZD6244 (ARRY-142886) is a potent, selective, orally-available, and non-ATP competitive small molecule inhibitor of the mitogen-activated protein (MAP) kinase kinase, MEK-1/2 (Investigator's Brochure, 2009; Friday and Adjei, 2008). AZD6244 inhibited the activity of purified MEK enzyme with an IC50 of 10-14 nM, and was found to be inactive or only A b brevi ate d Title: M olec ul ar Pr ofili n g N S C L C  
Versi o n D ate : 0 1/ 0 7/ 2 0 2 0 mi ni mall y acti ve at 1 0 M a gai nst a pa nel of ot h er ki nases, i ncl u di n g e pi der mal gr o wt h fact or rece pt or ( E G F R), E R B 2, p 3 8 , E R K2, a n d M K K 6 ki nases. Beca us e E R K is t he o nl y k n o w n s u bstrate of M E K, t he i n hi biti o n of M E K will tar get o nl y t he E R K si g nal tra ns d ucti o n pat h wa y a n d ot her si g nal tra ns d u cti o n pat h wa ys will n ot be bl oc ke d. A Z D 6 2 4 4 is m eta b olize d t o bi ol o gicall y acti ve N - des met h yl A Z D 6 2 4 4 w hic h is m ore p ote nt t ha n t he p are nt c o m p o u n d. I n vitr o, i n vi v o a n d preli mi nar y res ults fr o m cli nical st u dies s u g gest t hat A Z D 6 2 4 4 e x hi bits a fa v ora bl e p har mac ol o gic a n d t o xic ol o gic pr ofile.  
T he R A S/ R A F/ M E K/ E R K si g nali n g pat h w a y pla ys a ce ntr al r ole i n t he r e g ulati o n of man y cell ular pr o cesses i ncl u di n g pr olif erati o n, s ur vi val, differe ntiati o n, a p o pt osis, m otilit y, a n d meta b olis m ( O' N eill a n d K olc h, 2 0 0 4; Well br oc k et al., 2 0 0 4). T his pat h w a y is o ne of t he m ost i m p orta nt a n d best u n derst o o d M A P ki nase si g nal tra ns d ucti o n pat h wa ys, acti vate d b y a di vers e gr o u p of e xtracell ular si g nals i ncl u di n g i nte gri ns, gr o wt h fact or rece pt ors (i.e., E G F R, platel etderi ve d gr o wt h fact or rece pt or [ P D G F R], a n d i ns uli n-li ke gr o wt h f act or- 1 r ece pt or), a n d c yt o ki nes (J a nsse n et al., 2 0 0 5). Acti vate d R A S tri g gers t h e p h os p h or ylati o n a n d acti vati o n of R A F ki nase w hic h t h e n p h os p h or ylat es M E K 1 a n d M E K 2 o n 2 seri ne r esi d ues ( A h n et al.,  
2 0 0 1). Acti vate d M E K p h os p h or ylat es its o nl y k n o w n s u bstrates, E R K 1 a n d E R K 2.  
P h os p h or ylate d E R K di merizes a n d tra nsl ocat es t o t he n ucle us ( K h o k hlat c he v et al., 1 9 9 8) w her e it is i n v ol ve d i n se veral i m p orta nt cell ular f u n cti o ns, i ncl u di n g cell pr olif er ati o n.  
O vere x pressi o n of gr o wt h fact ors or gr o wt h fact or rece pt ors i n v ol ve d i n t he R A S/ R A F/ M E K/ E R K pat h wa y a n d act i vati n g ge n etic m utati o ns of t he si g n ali n g pr otei ns ma y lea d t o u nc o ntr olle d pr oliferati o n a n d t u m or f or mati o n. F or e x a m ple, R A S ge n es ar e t he m ost fre q ue ntl y m utate d o n c o ge n es detecte d i n h u ma n t u m ors (J a nsse n et al., 2 0 0 5). R A S pr otei ns are g ua ni n e n ucle oti de bi n di n g pr otei ns t hat acti vate R A F pr otei ns w he n b o u n d t o G T P. Ca ncerass ociate d m utati o ns i n R A S pr otei ns sta bilize t he G T P - b o u n d f or m of R A S, t here b y pr o vi di n g a c o nstit uti ve si g nal d o w nstrea m i n t he cas ca de. I n a d diti o n t o bei n g f o u n d i n al m os t all pa ncreatic a d e n ocarci n o mas, R A S m utati o ns are f o u n d i n ~ 5 0 % of c ol or ectal car ci n o mas, 2 55 0 % of l u n g a de n o car ci n o mas, a n d als o i n s o me breast or o varia n ca ncers. B R A F m utati o ns ha ve als o bee n o bser v e d i n ma n y h u ma n ca ncers, partic ularl y mela n o ma ( 3 0- 6 0 %), t h yr oi d ( 3 05 0 %), c ol orectal ( 5- 2 0 %), a n d o varia n ( ~ 3 0 %) ca n cers ( Well br oc k et al., 2 0 0 4; Fri da y et al.,  
2 0 0 8). T hese m utati o ns i n B R A F us u all y i n v ol ve gai n- of -f u n cti o n s u bstit uti o ns t hat re n der t he ki nases c o nstit uti vel y acti ve. Als o, st u dies of pri mar y t u m or s a m ples a n d cell li nes ha ve s h o w n c o nstit uti ve acti vati o n or o veracti vati o n of t h e M A P ki nase pat h wa ys i n ca n cers of t he p a ncreas, c ol o n, l u n g, o var y, a n d ki d ne y ( H os hi n o et al., 1 9 9 9). T heref ore, a ge nts tar geti n g t he R A S/ R A F/ M E K/ E R K pat h wa y m a y i n hi bit o nc o ge nic si g n ali n g i n t u m or cells.  
N o ncli nic al St u dies
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effic acy

I n vitr o st u dies ha v e s h o w n t hat A Z D 6 2 4 4 is p ote nt a n d selecti ve i n hi bit ors of M E K  
(I n vesti gat or's Br oc h ure, 2 0 0 9). H o we v er, si g nifica nt bi oc he mical acti vit y was n ot detecte d w he n teste d at 1 0 µ M a gai nst a di verse pa n el of 3 0 5 ot her m olec ules, i ncl u di n g e nz y m es, rece pt ors, ki nases, tra ns p orters, a n d i o n c h a n nels. T he eff ects of A Z D 6 2 4 4 o n E R K  
p h os p h or ylati o n a n d cell via bilit y wer e deter mi ne d i n a pa nel of cell li nes i n w hic h t he m utati o nal stat us of R A F a n d R A S are k n o w n. A Z D 6 2 4 4 i n hi bite d E R K 1 a n d E R K 2  
  
  
  
A b brevi ate d Title: M olec ul ar Pr ofili n g N S C L C  
Versi o n D ate : 0 1/ 0 7/ 2 0 2 0 p h os p h or ylati o n wit h I C 5 0 ra n gi n g fr o m 0. 0 0 1 8 t o 0. 0 4 1 M. A Z D 6 2 4 4 was partic ularl y p ot e nt i n i n hi biti n g t he via bilit y of cell li nes wit h V 6 0 0 E B R A F ge ne m utati o n a n d s o me cell li nes wit h K R A S m utati o ns. Se nsiti ve cell li nes i ncl u de d t h ose deri ve d fr o m c ol orectal, n o n- s mall cell l u n g ca ncer ( N S C L C), pa ncreatic, a n d mela n o ma t u m ors. T w o meta b olites of A Z D 6 2 4 4 ( Ndes met h yl A Z D 6 2 4 4 a n d a n a mi de A Z D 6 2 4 4) ha v e bee n i de ntifie d. T h e N des met h yl meta b olite was f o u n d t o be 3-t o 5-f ol d m ore acti v e t ha n t he par e nt c o m p o u n d i n cell ular E R K  
p h os p h or ylati o n i n hi biti o n assa ys, w her eas t he a mi de meta b olite was u p t o 5 0- f ol d less acti ve t ha n A Z D 6 2 4 4. I n t u m or cell via bilit y i n hi biti o n assa ys, N-des m et h yl met a b olite was ≥ 5 -f ol d m ore p ote nt i n i n hi biti n g cell via bilit y.  
Si g nifica nt s u p pressi o n of t u m or gr o wt h i n res p o nse t o A Z D 6 2 4 4 treat me nt was o bser v e d i n se veral x e n o graft m o use m o dels deri ve d fr o m a ra n ge of t u m or t y p es i ncl u di n g mela n o ma, breast, pa n creatic, l u n g, c ol o n, a n d he pat ocell ular carci n o mas. ( Tra n et al., 2 0 0 6; Brei kr e utz et al., 2 0 0 7; Da vies et al., 2 0 0 7; Tai et al., 2 0 0 7). I n pa pillar y t h yr oi d ca n cer m o dels, A Z D 6 2 4 4 effecti vel y i n hi bite d t u m or gr o wt h, b ot h i n vitr o a n d i n vi v o , partic ularl y i n t u m or cells carr yi n g acti vati n g B R A F ge ne m utati o ns ( Ball et al., 2 0 0 7). I n t he Cal u- 6 l u n g ca n cer x e n o graft m o del, A Z D 6 2 4 4 s u p presse d t u m or gr o wt h at d oses of 1 0, 2 5, or 1 0 0 m g/ k g gi ve n t wice dail y ( BI D),  
a n d t he mi ni mal effecti ve d ose was i de ntifie d as 0. 7 5 m g/ k g a d mi nistere d BI D ( Da vies et al.,  
2 0 0 7). I n t his m o del, M E K acti vit y w as i n hi bite d as assesse d b y d eter mi nati o n of p h os p h or ylat e d E R K ( p E R K) le vels i n t u m or. St u dies usi n g h u ma n c ol or ectal x e n o graft m o dels de m o nstrate d t hat A Z D 6 2 4 4 i n hi bite d t u m or gr o wt h b y i n hi biti o n of cell pr oliferati o n i n S W 6 2 0 m o del a n d b y i n d u cti o n of a p o pt osis i n C ol o 2 0 5 m o del. I n vi v o st u dies of t he K R A S m utati o np ositi ve ( K R A S +) h u ma n ca ncer x e n o grafts ha v e de m o nstrate d p ote ntial f or a d diti ve /s y n er gi stic effects of A Z D 6 2 4 4 i n c o m bi nati o n wit h a n u m ber of c yt ot o xic a n d tar get e d a ge nts, i ncl u di n g d oceta x el, iri n oteca n, ge mcita bi ne, Iress a ( gefiti ni b), A Z D 2 1 7 1 (ce dira ni b), ra pa m yci n, cis plati n, a n d te m oz ol o mi de (I n v esti gat or's Br o c h ure, 2 0 0 9).  
P h ar m ac oki netic/ P h ar m ac o dy n a mic St u dies T w o oral f or m ulati o ns of A Z D 6 2 4 4 ha ve b ee n test e d i n precli nical p h ar mac ol o g y st u dies: t he ori gi nal mi x a n d- dri n k f or m ulati o n ( A Z D 6 2 4 4 fr ee- base), a n d t he A Z D 6 2 4 4 h y dr o ge n s ulfate salt (ca ps ule) f or m ulati o n ( A Z D 6 2 4 4 H y d- S ulfat e) (I n v esti gat or's Br oc h ure, 2 0 0 9). T h e f or mer re q uires r ec o nstit uti o n of t he A Z D 6 2 4 4 fr ee- b ase cr ystalli ne p o w der i n t he 2 5 % ( w/ v) A q u e o us Ca ptis ol ®(s ulf o b ut yl et her β c ycl o d e xtri n) ( S B E - C D) s ol uti o n. A bs or pti o n of A Z D 6 2 4 4 fr eebase was m o derate t o hi g h at l o w d oses i n rat, d o g a n d m o n ke y, b ut s h o we d decreasi n g bi oa vaila bilit y wit h i ncreasi n g d ose. T he decli ni n g bi oa v aila bilit y at hi g her d oses ma y be d u e t o t he l o w a q ue o us s ol u bilit y of A Z D 6 2 4 4. W he n a d mi nisteri n g A Z D 6 2 4 4 h y dr o ge n s ulfate, A Z D 6 2 4 4 e x p os ure, e x presse d as a n area u n der t h e plas ma c o nce ntrati o n -ti me c ur ve fr o m 0 t o 1 2 h o urs ( A U C 0- 1 2), i ncrease d r o u g hl y i n pr o p orti o n wit h d ose i n t he m o use a n d m o n ke y. I n 1m o nt h st u dies, 3 t o 6-f ol d gr eater e x p os ures wer e ac hie ve d wit h A Z D 6 2 4 4 h y dr o ge n s ulfate at 7. 5 m g/ k g BI D t ha n wit h t he A Z D 6 2 4 4 fr ee- b ase at 3 0 m g/ k g BI D. T here was n o/ mi ni mal acc u m ulati o n of A Z D 6 2 4 4, w het her d ose d wit h A Z D 6 2 4 4 fr ee- b ase or A Z D 6 2 4 4 h y dr o ge n s ulfate, o n m ulti ple d osi n g i n t he m o use, r at, or m o n ke y. Oral bi oa v aila bilit y of A Z D 6 2 4 4 i n m o n ke y, f oll o wi n g d osi n g wit h 5 m g/ k g of eit her free- base or ca ps ul e, was 2 9 % a n d 5 6 %,  
res pecti vel y. N des met h yl meta b olite w as n ot detecta ble i n r at a n d at o nl y trace le v els i n t he m o n ke y, b ut w as pr o d uce d i n m o use at circ ul ati n g le v els ar o u n d 2- 1 2 % of pare nt c o m p o u n d.  
A b brevi ate d Title: M olec ul ar Pr ofili n g N S C L C  
Versi o n D ate : 0 1/ 0 7/ 2 0 2 0 St u dies i n rats i n dicate t hat A Z D 6 2 4 4 is wi del y distri b ute d, alt h o u g h tiss ue c o nce ntr ati o ns were l o wer t ha n bl o o d c o nce ntrati o ns. Hi g h le v els of pr otei n bi n di n g ( 9 3. 7- 9 9. 7 %) were o bser ve d i n all precli nical s pecies teste d a n d i n h u ma ns ( 9 8. 4 %). T her e was n o e vi de n ce of A Z D 6 2 4 4relate d material bi n di n g t o mela ni n a n d mi ni mal pe netrati o n i nt o t he ce ntr al ner v o us s yste m  
( C N S). A ZD 6 2 4 4 was meta b olize d b y c yt oc hr o me P 4 5 0 ( C Y P e nz y m es) 1 A 2, 2 C 1 9 a n d 3 A 4, wit h C Y P 1 A 2 bei n g t he e nz y me pri maril y r es p o nsi ble f or t he f or mati o n of t he N- des met h yl meta b olite. Gl uc ur o ni dati o n a p pears t o be a si g nifica nt cleara n ce mec ha nis m f or A Z D 6 2 4 4 a n d N- des met h yl meta b olite. A Z D 6 2 4 4 di d n ot i n hi bit C Y P 1 A 2, 2 C 8, 2 C 1 9, 2 D 6, a n d 3 A 4. It w as a wea k i n hi bit or of C Y P 2 C 9 (I C 5 0 4 4. 7 µ M). N- des met h yl A Z D 6 2 4 4 di d n ot i n hi bit C Y P 2 A 6, 2 C 8, 2 C 9, 2 C 1 9, 2 D 6, 2 E 1, or 3 A 4, b ut was a wea k i n hi bit or of 1 A 2 (I C 5 0 of 1 8. 9 µ M). I n rat, m o use a n d m o n ke y, fecal e x creti o n was t he pre d o mi na nt r o ute after oral a n d i ntra ve n o us d osi n g.  
T oxi c ol o gic St u dies T he t o xic ol o gic eff ects of A Z D 6 2 4 4 were e val uat e d i n ac ute d ose (si n gl e or t w o d oses o n a si n gle da y) a n d 1- m o nt h, re peat- d ose st u dies i n S pra g ue- Da wle y rats a n d c y n o m ol g us m o n ke ys  
(I n vesti gat or's Br oc h ure, 2 0 0 9). T he repeat d os e st u d y i n rats i n dicat e d t hat t he a ge nt was well t olerate d b ut pr o d uce d s oft st o ols a n d gastr oi ntesti nal m uc osal mi neralizati o n ass ociate d wit h i ncreas e d ser u m p h os p h or us a n d decreas e d al b u mi n. Diarr hea, de h y drati o n, electr ol yte i m bala nce, a n d s ec o n dar y r e nal c h a n ges w ere o bs er ve d i n m o n ke ys. T he v e hicle, Ca ptis ol ®,  
use d t o rec o nstit ute A Z D 6 2 4 4 free- base p o w der, is k n o w n t o ca use gastr oi ntesti nal dist ur ba nces i n precli nical st u dies. A re d ucti o n i n t he v ol u me of Ca ptis ol ® a d mi nistrati o n t o m o n ke ys w as ass ociate d wit h a r e d uce d i nci de nce of di arr h ea; h o we ver, it is li kel y t h at A Z D 6 2 4 4 e x acer b ate d t he ve hicle effects. T he n o o bser va ble a d vers e effect le vels ( N O A E Ls) i n rats (fe m ale) a n d m o n ke ys i n 1- m o nt h st u dies were i d e ntifie d at 1 0 m g/ k g/ da y BI D ; a N O A E L w as n ot ac hie v e d i n male rats. D osi n g wit h A Z D 6 2 4 4 h y dr o ge n s ulfate ( 0. 5, 1. 5, a n d 4 m g/ k g BI D) f or u p t o 6 m o nt hs t o m o n ke ys was als o ass ociate d wit h fl ui d a n d/ or re d- c ol ore d feces, b ut wit h n o n ota ble gastr oi ntesti nal tr act or r e nal pat h ol o g y. Tiss ue mi neralizati o n was n ot a p pare nt i n c y n o m ol g us m o n ke ys d ose d f or u p t o 6 m o nt hs wit h A Z D 6 2 4 4 h y dr o ge n s ulfate. H o we ver, mi neralizati o n was see n i n m ulti ple tiss ues (c or n ea, ki d ne y, li ver, m y ocar di u m, s keletal m uscle, gl a n d ular st o mac h) i n mice d ose d wit h A Z D 6 2 4 4 h y dr o ge n s ulfate f or u p t o 1 m o nt h, a n d i n t he li vers of a s mall n u m ber of mice d ose d u p t o 6 m o nt hs.  
A Z D 6 2 4 4 a n d its N- des met h yl meta b olite s h o we d n o e vi de nce of m uta ge nic p ote ntial, b ut A Z D 6 2 4 4 pr o d uce d a n i ncreas e i n micr o n ucleate d i m m at ure er yt hr oc yt es i n mice, pre d o mi na ntl y via a n a n e u ge ni c m o de of acti o n. A Z D 6 2 4 4 s h o we d e n h a n ce d c yt ot o xicit y i n t he prese nce of ultra vi olet ( U V) li g ht. Wit h t he e x ce pti o n of tiss ue mi neralizati o n i n rats a n d mice, t here was e vi de nce of r e v ersi bilit y of m ost c ha n ges i n t he 1- or 6 - m o nt h st u dies wit h A Z D 6 2 4 4  
(free base or h y dr o ge n s ulfate). Re pr o d u cti ve t o xic ol o g y d ata i n mice i n dicate t hat A Z D 6 2 4 4 ca n ha v e a d verse effects o n e m br y ofetal d e vel o p me nt a n d s ur vi val at d ose le vels t hat wer e n ot t o xic t o m others.  
I n s u m mar y, precli nical st u dies de m o nstrate d t hat A Z D 6 2 4 4 e x p os ures ca n be si g nifica ntl y e n ha nce d b y usi n g A Z D 6 2 4 4 h y dr o ge n s ulfate, c o m pare d t o A Z D 6 2 4 4 free- base. I n 6- m o nt h t o xic ol o g y st u dies, A Z D 6 2 4 4 e x p os ures ( A U C 0- 1 2) ac hie v e d wit h A Z D 6 2 4 4 h y dr o ge n s ulfate at hi g h d ose le v els ( 4 m g/ k g BI D i n m o n ke ys a n d 2 0 m g/ k g BI D i n mice) were a p pr o xi matel y 3-  
fold and 15-fold higher in primates and mice, respectively, when compared with those achieved in man at 75 mg BID AZD6244 hydrogen sulfate. At the NOAEL of 1.5 mg/kg BID from 1month and 6-month studies in primates, exposures were generally similar to those seen in man after dosing at 75 mg BID AZD6244 hydrogen sulfate. Exposure achieved in the 6-month mouse study at the low dose level of 1 mg/kg BID was slightly below that seen to date in man after dosing with AZD6244 hydrogen sulfate (75 mg BID). Preclinical pharmacology studies suggest an acceptable safety profile for the administration of AZD6244 free base or AZD6244 hydrogen sulfate to human cancer patients. Clinical Experience V
AstraZeneca sponsored a phase 1 open-label study designed to assess the safety, tolerability and pharmacokinetics (PK) of AZD6244 hydrogen sulfatein patients with advanced solid malignancies. In addition, this study planned to assess the relative bioavailability of the capsule vs. free-base formulation. This is the first clinical study that employed the AZD6244 capsule formulation. The further development of AZD6244 is to be continued with the hydrogen sulfate salt (capsule formulation) (Investigator's Brochure, 2009).  
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This study was conducted in two parts. Part A (31 patients enrolled) of the study was a dose escalation study, was designed to provide adequate tolerability, safety, PK, and pharmacodynamic data. In Part A, the first cohort received a single 25 mg dose of the AZD6244 hydrogen sulfate on day 1, followed by BID dosing from day 2 onwards. Other doses investigated were 50 mg, 75 mg, and 100 mg. The aim of Part B (29 patients randomized) was to determine the relative oral bioavailability of the AZD6244 hydrogen sulfate, and secondly to expand the safety, tolerability and preliminary efficacy data for the maximum tolerated dose (MTD) of 75 mg BID. In Part B, patients received either a single dose of capsule formulation or free-base suspension formulation on day 1 and 8, followed by continuous BID dosing of capsule formulation from day 9 onwards.  
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An AstraZeneca-sponsored multi-arm combination study of the AZD6244 capsule formulation that evaluates PK interactions of AZD6244 with selected chemotherapy agents (docetaxel, dacarbazine, erlotinib, temsirolimus) is ongoing.
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pharmacokinetics

AZD6244 plasma PK parameters of AZD6244 hydrogen sulfate were similar after single and multiple dosing, suggesting the minimal accumulation over time after BID dosing. The AZD6244 exposure parameters, i.e., the maximum plasma concentration (Cmax) and the area under time-concentration curve (AUC) were approximately dose proportional across the 25- to 100-mg BID dose range after single (day 1) or multiple dosing studied on days 1, 8, 15, and 22: the geometric mean values of exposure parameters were: Cmax=369-1489 ng/mL and AUC=1361-7055 ng∙h/mL on day 1, and Cmax=458-1365 ng/mL and AUC0-12=1515-4758 ng∙h/mL on day 8. AZD6244 was absorbed relatively quickly across all dose levels, with a median time-to-reach Cmax (tmax) of 1.5 hours. Following the peak, AZD6244 concentration declined multi-exponentially with a mean terminal elimination half-life (t1/2) ranging from 5 to 7 hours, which was consistent across dose levels. The steady state volume of distribution (Vss) and clearance (Cl) also remained largely consistent across the dose range, with mean values D  
ranging from 87 to 126 L and 12 to 19 L/h, respectively. Plasma concentrations of N-desmethyl AZD6244 followed a similar PK profile as that of AZD6244, although exposure was much lower, achieving Cmax and AUC values generally <15% of the parent compound, in each patient. The median tmax was approximately 1.5 hours and t1/2 ranged from 9-13 hours. The AZD6244 amide metabolite showed increased exposure on multiple dosing indicating some accumulation. Concentrations of the AZD6244 amide varied greatly among patients and t1/2 could not be assessed. Given the 3-to 5-fold greater potency of the N-desmethyl metabolite compared to AZD6244 shown by the in vitro cell-based ERK phosphorylation assay, the former is likely to contribute to pharmacodynamic effects. In contrast, AZD6244 amide, which was approximately 40- to 50-fold less active than AZD6244 in this assay, is unlikely to contribute significantly to AZD6244 biological activity. Nevertheless, both metabolites will be measured in all future AstraZeneca-sponsored clinical studies of AZD6244 hydrogen sulfate.  
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The capsule formulation was demonstrated to significantly improve the oral bioavailability, although large inter-patient variability was noted. The estimated oral bioavailability of capsule relative to the free-base suspension based on a dose-normalized AUC0-24 was 263% (90% CI = 214 to 322%). The geometric mean values of the exposure parameters obtained for AZD6244 hydrogen sulfate at 75 mg BID (MTD) were: AUC0-24 = 6335 and 5448 ng∙h/mL on day 1 and 8, respectively, and Cmax = 1207 and 1439 ng/mL on day 1 and 8, respectively). In comparison, the exposure achieved by hydrogen sulfate at the MTD was significantly higher than that achieved by the free base suspension at 100 mg BID (MTD); the estimated exposures by hydrogen sulfate relative to those by free-base, achieved at their respective MTDs, were 197% (90% CI = 161 to 242%) and 252% (90% CI = 182 to 348%) based on AUC0–24 and Cmax, respectively.  
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A food effect study involving administration of AZD6244 hydrogen sulfate to patients with advanced solid malignancies under fasting conditions and with a high-fat meal indicated a statistically significant effect of food on the exposure of AZD6244. Geometric mean Cmax and AUC values were reduced by approximately 60% and 20%, respectively, under fed conditions. Therefore, it is recommended for further clinical studies that AZD6244 should continue to be taken on an empty stomach (no food or drink other than water for 1 hour prior to dosing and 2 hours after dosing).  
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There is no evidence of a PK interaction between AZD6244 and either docetaxel or dacarbazine, when given in combination as either twice daily dosing of AZD6244 50 mg or 75 mg with docetaxel 75 mg/m2 on day 1 of a 21 day cycle, or as twice daily dosing of AZD6244 50 mg or 75 mg with dacarbazine 1000 mg/m2 on day 1 of a 21 day cycle. Pharmacodynamics D
Inhibition of phosphorylation of ERK as an indirect measure of MEK inhibition by AZD6244 hydrogen sulfate was assessed in PBMCs from each patient at 4 time-points post-single-dose administration, across the dose levels (25-100 mg). Inhibition of ERK phosphorylation was observed at all doses, with the greatest inhibition generally occurring at the first time point (1hour post dose); 73% inhibition was seen at 1-hour at the 75-mg BID dose, which declined to ~23% at 8-hour time-point.
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clinical activity

In the AZD6244 hydrogen sulfate single-agent study, 55 patients had RECIST evaluable tumors. There was one complete response in the 75-mg dose cohort in Part A after 16 weeks. This patient had a BRAF+ melanoma. At the 75-mg dose, 16/35 (45.7%) patients had stable disease for ≥6 weeks. Nine patients in Part A and 13 patients in Part B had a best response of stable disease. Ten out of 55 (18.2%) patients (not including the patient who had a complete response) had stable disease of ≥16 weeks. Safety V
Twenty-eight patients were dosed in Part A of the study: 25 mg BID (n=6), 50 mg BID (n=7), 75 mg BID (n=7) and 100 mg BID (n=8). One dose-limiting toxicity (DLT) (grade 3 fatigue that resolved on discontinuation of AZD6244 hydrogen sulfate) was reported in the first 22 days of dosing in the 75 mg BID cohort. Two DLTs were reported in the first 22 days of dosing in the 100 mg BID cohort: grade 3 pleural effusion and grade 3 rash.  
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Based on the data from this study, the emerging safety and tolerability profiles for the AZD6244 capsule formulation are broadly consistent with both the patient population, and the adverse event profile seen with the AZD6244 free-base suspension formulation. The most frequently reported adverse events (AEs), regardless of dose cohort, severity, causality, or seriousness, were fatigue, acneiform dermatitis, nausea, diarrhea, and peripheral edema. All AEs of acneiform dermatitis were considered treatment related in all dose cohorts. In patients receiving 75 mg BID, all fatigue events were considered treatment related. Twenty-one patients reported at least one serious AE. Seven patients had at least one serious, treatment-related AE, with only hypertension being reported on more than one occasion. Thirty-three of 56 patients (58.9%) reported at least one grade 3 AEs during the study, of whom 18/56 (32.1%) experienced at least one treatment-related AE of ≥grade 3. Eighteen patients had permanent discontinuation of study treatment due to at least one AE (fatigue, anorexia, vomiting, nausea, left ventricular (LV) dysfunction, visual disturbance, fungal urinary tract infection, and acute renal failure). None of the 13 deaths occurred in this study was attributed to the therapy agent. Comparison of the AE frequencies from the AZD6244 hydrogen sulfate study and phase 2 monotherapy studies of AZD6244 free-base shows higher proportion of patients reporting the most frequent AEs, such as fatigue, dermatitis acneiform, diarrhea, nausea, and peripheral edema, with the capsule formulation. This may be due to the higher plasma exposures achieved with the capsule formulation, but may also be in part a consequence of more heavily pretreated patient population in the single-agent AZD6244 hydrogen sulfate (capsule) study. A higher proportion of patients withdrew due to AE at the MTD level (75 mg BID in the AZD6244 hydrogen sulfate study than at the AZD6244 free-base MTD (100 mg BID), although the different patient populations may be a contributing factor.  
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