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1 LBx Liquid Biopsy

2 LBx Liquid Biopsy Test Report Patient ID: CM_01 Report ID: CellMax Life 1 Report Date: Dec 15, 2015 Client ID: POC SNOMED Concept Id: Disease: Carcinoma of colon (disorder) Table of Contents 1. Summary 2. Complete List of Biomarkers Detected 3.1. Details on Biomarkers Detected 3.2. Details on Biomarkers Detected 3.3. Details on Biomarkers Detected 3.4. Details on Biomarkers Detected 3.5. Details on Biomarkers Detected 3.6. Details on Biomarkers Detected 4. References 5. Glossary 1

3 LBx Liquid Biopsy Test Report Patient ID: CM_01 Report ID: CellMax Life 1 Report Date: Dec 15, 2015 Client ID: POC SNOMED Concept Id: Disease: Carcinoma of colon (disorder) 1 Summary 2

4 LBx Liquid Biopsy First Patient Last Name 1271 Oakmead Parkway Sunnyvale, CA 94085, USA 3

5 LBx Liquid Biopsy SUMMARY RESULT: POSITIVE Clinically Relevant Genomic Alterations Detected Marker Biological Test Association Result Therapies approved in Colorectal carcinoma Therapies approved in other indications May indicate resistance to therapies Cetuximab, Panitumumab KRAS KRAS MUTN (seq) G13D None Cobimetinib, Trametinib Yes APC APC MUTN (seq) E1536* None Celecoxib No Yes AKT1 PI3K/Akt/m MUTN (seq) TOR E17K None Everolimus, Temsirolimus No Yes TP53 p53 MUTN (seq) R175H None None No Yes Trials Lab Supervisor Date Pathologist Date M 1271 Oakmead Parkway Sunnyvale, CA 94085, USA 4

6 LBx Liquid Biopsy Test Report Patient ID: CM_01 Report ID: CellMax Life 1 Report Date: Dec 15, 2015 Client ID: POC SNOMED Concept Id: Disease: Carcinoma of colon (disorder) 2 Complete List of Biomarkers Detected 5

7 LBx Liquid Biopsy Test Report Patient ID: CM_01 Report ID: CellMax Life 1 Report Date: Dec 15, 2015 Client ID: POC SNOMED Concept Id: Disease: Carcinoma of colon (disorder) 1.1 POSITIVE BIOMARKERS Marker Biological Association Test Result Therapies approved in Colorectal carcinoma Therapies approved in other indications May indicate resistance to therapies Cetuximab, Panitumumab KRAS KRAS MUTN (seq) G13D None Cobimetinib, Trametinib Yes APC APC MUTN (seq) E1536* None Celecoxib No Yes AKT1 PI3K/Akt/m MUTN (seq) TOR E17K None Everolimus, Temsirolimus No Yes TP53 p53 MUTN (seq) R175H None None No Yes 1.2 VARIANTS OF UNCERTAIN CLINICAL SIGNIFICANCE Marker Biological Association Test Result MLH1 HNPCC MUTN (seq) V384D DNMT3A Epigenetic regulation MUTN (seq) C911Y The functional or therapeutic consequences of VARIANTS OF UNCERTAIN CLINICAL SIGNIFICANCE are unknown. 1.3 LABORATORY TECHNICAL DATA Variant Map Location Coding Sequence Change KRAS-G13D chr12: c.38g>a APC-E1536* chr5: c.4606g>t AKT1-E17K chr14: c.49g>a TP53-R175H chr17: c.524g>a MLH1-V384D chr3: c.428t>a DNMT3A-C911Y chr2: c.2732g>a The data in this table was generated by the laboratory in the course of molecular testing. It has not been altered in any way by N-of- One. 2 Guidelines Marker KRAS Summary For colorectal carcinoma patients with metastatic disease and tumors harboring a KRAS or NRAS mutation, the NCCN guidelines (v ) recommend against the use of cetuximab and panitumumab. Trials 6

8 3 Actionable Biomarkers/Pathways 3.1 KRAS BIOMARKER RESULTS SUMMARY Marker Result Summary KRAS - MUTN (seq): G13D KRAS-G13D is an activating mutation. KRAS encodes the signaling protein K-Ras, a member of the Ras family; activating KRAS alterations may result in activation of downstream signaling pathways, including the Raf/MEK/ERK pathway (Pylayeva-Gupta et al., 2011; , Nakano et al., 1984; ). Several MEK inhibitors are under clinical investigation, including the FDA-approved therapies trametinib and cobimetinib, and may be relevant for tumors harboring K-Ras activation (Flaherty et al., 2012; , Larkin et al., 2014; , Britten, 2013; , Jänne et al., 2013; ). For colorectal carcinoma patients with metastatic disease and tumors harboring a KRAS or NRAS mutation, the NCCN guidelines (v ) recommend against the use of cetuximab and panitumumab BIOLOGICAL RELEVANCE of KRAS KRAS alterations in Colorectal carcinoma Molecular function Incidence in disease The KRAS G13D mutation lies within the first "G box" domain of the K-Ras protein, one of several conserved regions responsible for GTP binding and hydrolysis (Colicelli, 2004; ). Mutation of the adjacent codon 12 creates a protein that is defective for GTP hydrolysis and is therefore constitutively active (Jackson et al., 2001; ). While KRAS codon 13 mutations, including G13D, have been reported to be activating, cells transformed with a codon 13 mutation grew less aggressively than cells transformed with KRAS codon 12 mutants (Guerrero et al., 2000; , Alamo et al., 2015; ). KRAS mutations have been reported in 36% (3943/10875) of colorectal carcinoma samples analyzed in COSMIC (Aug 2015) and in 42% (94/224) of sequenced tumors in the Colorectal Adenocarcinoma TCGA/Nature dataset (COSMIC, cbioportal for Cancer Genomics, Aug 2015). KRAS mutations have been reported in 30-40% of colorectal cancer samples analyzed in scientific studies; however, many studies have only assayed for KRAS mutational hotspots, such as codons 12 and 13 (Gil et al., 2014; , Xie et al., 2015; , Peeters et al., 2013; , Maus et al., 2014; , Li et al., 2015; , Paliogiannis et al., 2014; ). 7

9 LBx Liquid Biopsy Test Report Patient ID: CM_01 Report ID: CellMax Life 1 Report Date: Dec 15, 2015 Client ID: POC SNOMED Concept Id: Disease: Carcinoma of colon (disorder) 3.1 Details on Biomarkers Detected 8

10 3.1.3 CLINICAL RELEVANCE of KRAS KRAS alterations in Colorectal carcinoma Role in disease Effect on drug sensitivity Effect on drug resistance The KRAS gene is one of the most commonly mutated genes in human malignancies, with high incidences in pancreatic, colorectal, and lung cancers (Farber et al., 2011; , Feldmann et al., 2007; , Han et al., 2011; ). Activating KRAS mutations can result in constitutive activation of the Ras/Raf/MEK/ERK and PI3K/Akt pathways (Nakano et al., 1984; , Pylayeva-Gupta et al., 2011; ). Studies using xenograft models or cell lines of colorectal cancer have reported that activating KRAS mutations, often assessed in the context of other genetic alterations, can play a role in the development and progression of colorectal cancer (Davies et al., 2014; , Cagnol and Rivard, 2013; , Trobridge et al., 2009; ). Many of the current attempts to target K-Ras are directed against its downstream signaling pathways, Raf/MEK/ERK and PI3K/Akt/mTOR (Yeh et al., 2009; , Britten, 2013; ). The MEK inhibitors trametinib and cobimetinib (in combination with vemurafenib) have been FDA-approved for BRAF V600E- and V600K-mutant melanoma and are under investigation in clinical trials (Flaherty et al., 2012; , Larkin et al., 2014; ). A novel clinical approach for KRAS-positive tumors, based on synthetic lethal interactions that occur in the presence of a KRAS mutation and either diminished Cdk4 activity or diminished Bcl-2/ Bcl-xL activity, is a treatment combination of MEK inhibition and either Cdk4/6 inhibition or Bcl-2/Bcl-xL inhibition (Mao et al., 2014; , Puyol et al., 2010; , Tan et al., 2013; , Corcoran et al., 2013; ). In some cancer types, such as colorectal cancer and non-small cell lung cancer, activating KRAS mutations and KRAS amplification have been associated with resistance to Egfr-targeted therapies (Eberhard et al., 2005; , Linardou et al., 2008; , Ramos et al., 2008; , Campos-Parra et al., 2013; , De Roock et al., 2011; , Douillard et al., 2013; , Valtorta et al., 2013; , Li et al., 2014; ). Current NCCN guidelines (2014) recommend against the use of cetuximab or panitumumab in colorectal cancer patients with any known KRAS mutation (NCCN.org). Several preclinical and clinical studies have suggested that colorectal tumors with KRAS G13D mutations may be sensitive to the Egfr inhibitors cetuximab or panitumumab; however, other studies have reported no significant effect of cetuximab in colorectal cancer patients with KRAS G13D mutations (Kumar et al., 2014; , Messner et al., 2013; , Waring et al., 2014; ASCO 2014, Abstract e14536, De Roock et al., 2010; , Tveit et al., 2012; , Schirripa et al., 2015; ). In addition, KRAS mutant allele-specific imbalance (MASI) has been reported in 12.8% (58/436) of metastatic colorectal cancer samples, and was particularly associated with the G13D mutation. There was no statistically significant change in survival between MASI negative and positive patients. A cell line with KRAS G13D MASI exhibited resistance to cetuximab (Malapelle et al., 2015; ). 9

11 3.1.4 CLINICAL EVIDENCE in Colorectal carcinoma KRAS alterations in Colorectal carcinoma FDA Approved Phase III Data Phase II Data Phase I Data Preclinical None. A large retrospective study found that response to the Egfr inhibitor antibody cetuximab (Erbitux ) was significantly better in KRAS wild-type tumors compared to KRAS-mutant tumors in colorectal cancer (n = 605) (De Roock et al., 2010; ). Large, randomized Phase 3 trials in colorectal cancer have found that panitumumab, when combined with chemotherapy, results in prolonged progression-free survival compared to chemotherapy alone in patients with wild-type KRAS, but not in patients with mutant KRAS (Douillard et al., 2010; , Peeters et al., 2010; , Douillard et al., 2014; , Peeters et al., 2014; ). A Phase 2 trial of selumetinib in patients with KRAS-mutant colorectal carcinoma who were progressing on first-line therapy reported partial response and stable disease lasting at least four weeks in 9.7% (3/31) and 51.6% (16/31) of patients, respectively; notably, three patients were reported to have stable disease lasting more than one year (Hochster et al., 2014; ). A Phase 2 clinical trial of temsirolimus as a single agent or in combination with irinotecan in patients with KRAS-mutant CRC has reported stable disease in 38% (24/64) and 63% (40/63) of patients on monotherapy or combination therapy, respectively; however, all patients who exhibited tumor reduction were found to have low levels of mutated KRAS in plasma samples (Spindler et al., 2013; ). A Phase 2 study which compared selumetinib with capecitabine as monotherapy in colorectal cancer patients reported stable disease in 29% (10/34) of patients in the selumetinib group as compared to 43% (15/35) in the capecitabine group. No significant differences in progression-free survival times or in the number of patients with disease progression were reported (Bennouna et al., 2011; ). A Phase 1 dose-escalation study of binimetinib in pretreated patients with advanced solid tumors has reported partial response in 6% (1/17) of patients and stable disease in 53% (9/17) of patients; the authors report that binimetinib displayed an acceptable safety profile and induced pharmacodynamics changes suggestive of MEK inhibition (Bendell et al., 2011; AACR-NCI-EORTC 2011, Abstract B243). In a Phase 1 trial of the MEK inhibitor trametinib in patients with advanced solid tumors, an overall 10% objective response rate was reported. No objective responses were seen in 28 patients with colorectal cancer, although nine patients had stable disease (Infante et al., 2012; ). A study of PD in 13 patients with metastatic tumors (seven melanoma, three breast, and three colorectal cases) reported clinical benefit in six patients (one confirmed complete response in a melanoma case as well as five patients with stable disease). However, the drug was not well tolerated at the doses administered and the study was terminated early (Boasberg et al., 2011; ). A Phase 1 multicenter trial of refametinib in patients with advanced cancer reported suppression of ERK phosphorylation and stable disease in 11 patients for four or more courses of therapy (Weekes et al., 2013; ). A Phase 1b trial of the combination of copanlisib (BAY ), a pan-pi3k inhibitor, and refametinib in patients with advanced cancer has reported a partial response in one endometrial cancer patient and stable disease in nine patients (Ramesh et al.,; ASCO 2014, Abstract 2588). A Phase 1 study of cobimetinib in combination with pictilisib in patients with advanced solid tumors reported partial responses and stable disease for at least five months in 3/46 (a melanoma patient with mutated BRAF, a pancreatic carcinoma patient with mutated BRAF, and an endometrioid carcinoma patient with mutated KRAS) and 5/46 evaluable patients, respectively (LoRusso et al., 2012; ASCO 2012, Abstract 2566). A Phase 1 study of cobimetinib in 13 patients with advanced solid tumors reported that treatment was well tolerated; one patient with progressive NSCLC exhibited ongoing stable disease of at least seven months (Rosen et al., 2008; ASCO 2008, Abstract 14585). Studies in cell lines suggest that KRAS mutations may predict response to downstream MEK inhibition in CRC (Yeh et al., 2009; ). Combination treatment with a PI3K inhibitor BKM120 and MEK inhibitor PD was reported to induce tumor regression in a mouse model of PIK3CA wild-type, KRAS mutant colorectal cancer (Roper et al., 2014; ). KRAS mutant colorectal cancer cells show selective vulnerability to combined treatment with proteasome inhibitors and DNA-damaging agents in preclinical studies 10

12 (Steckel et al., 2012; ). A preclinical study reported that lenvatinib inhibited tumor growth and reduced tumor-associated vessel density in KRAS mutant colorectal carcinoma xenograft models (Wiegering et al., 2014; ). A preclinical study reported that, whereas KRAS G12V-mutant CRC cells were resistant to the EGFR inhibitor cetuximab, cell proliferation in the cells was inhibited by cetuximab in combination with the HIF-1alpha inhibitor PX-478 (Wang et al., 2015; ). A preclinical study reported that refametinib inhibited tumor growth in colon cancer xenograft models (Iverson et al., 2009; ) SAMPLE RELEVANT THERAPIES Therapies targeting MEK Drug Trade Name Target/Rationale Current Status Trametinib Mekinist MEK1,2 inhibitor. Phase 2 (Colorectal carcinoma) FDA Approved (Melanoma) Cobimetinib Cotellic MEK1 inhibitor. Phase 1 (Solid Tumor) FDA Approved (Melanoma) Selumetinib MEK1,2 inhibitor. Phase 2 (Colorectal carcinoma) Phase 3 (Melanoma, Non-small cell lung carcinoma (NSCLC), Thyroid carcinoma) Binimetinib MEK1,2 inhibitor. Phase 2 (Colorectal carcinoma) Phase 3 (Melanoma, Ovarian carcinoma) Refametinib MEK1 inhibitor. Phase 2 (Colorectal carcinoma) Phase 2 (Gallbladder carcinoma, Hepatocellular carcinoma, Pancreatic carcinoma, Breast carcinoma, Cholangiocarcinoma) Pimasertib MEK1,2 inhibitor. Phase 2 (Colorectal carcinoma) Phase 2 (Melanoma, Pancreatic ductal adenocarcinoma, Hematologic malignancies) PD MEK1,2 inhibitor. Phase 2 (Colorectal carcinoma) Phase 2 (Pancreatic carcinoma, Melanoma, Non-small cell lung carcinoma (NSCLC), Breast carcinoma, Neurofibromatosis type 1) Therapies targeting Raf Drug Trade Name Target/Rationale Current Status Defactinib second-generation FAK and Pyk2 inhibitor. Phase 1 (Solid Tumor) Phase 2 (Mesothelioma) 11

13 3.1.6 BIOMARKER-MATCHED CLINICAL TRIALS Trials Prioritized By Clinical Specificity* Markers TrialID Title Phase Targets Locations/contact 1 KRAS NCT Trametinib and Navitoclax in Treating Patients With Advanced or Metastatic Solid Tumors 2 KRAS NCT A Study of MEHD7945A and Cobimetinib (GDC-0973) in Patients With Locally Advanced or Metastatic Cancers With Mutant KRAS 3 KRAS NCT Selumetinib and Cyclosporine in Treating Patients With Advanced Solid Tumors or Advanced or Metastatic Colorectal Cancer 4 KRAS, AKT1, TP53 NCT NCI-MPACT: Molecular Profiling-Based Assignment of Cancer Therapy for Patients With Advanced Solid Tumors 5 KRAS NCT A Phase Ib Study of MEK162 Plus BYL719 in Adult Patients With Selected Advanced Solid Tumors Phase 1/Phase 2 Phase 1 MEK Phase 1 MEK MEK, Bcl- 2, BCL2L2 Phase 2 MTOR, PARP, Wee1, MEK Phase 1/Phase 2 *Note The trials listed above were matched to the biomarkers identified in this tumor; the list may not be comprehensive. Dana-Farber Cancer Institute: Massachusetts, USA, Geoffrey I. Shapiro, geoffrey_shapiro@dfci.harvard.edu, (MA) Massachusetts General Hospital Cancer Center: Massachusetts, USA, Ryan B. Corcoran, rbcorcoran@partners.org, (MA) Overall contact: Reference Study ID Number: GO _worldwide.htm, global.rochegenentechtrials@roche. com, (U.S. Only) CA (1), CO (1), CT (1), TN (1), TX (1) CO (1), MO (2), NC (1), NJ (1), OH (1), PA (1), TX (1) Overall contact: Jennifer H Zlott, zlottjh@mail.nih.gov, (301) National Institutes of Health Clinical Center, 9000 Rockville Pike: Maryland, USA, For more information at the NIH Clinical Center contact National Cancer Institute Referral Office, (MD) MEK, PI3K Overall contact: Array BioPharma Clinical Trial Call Center, CA (1), FL (1), IL (1), MA (1), NY (2), UT (1), Barcelona (2), Bellinzona (1), Parkville (1) 12

14 Trials Prioritized By Region* Markers TrialID Title Phase Targets Locations/contact 1 KRAS NCT A Study of MEHD7945A and Cobimetinib (GDC-0973) in Patients With Locally Advanced or Metastatic Cancers With Mutant KRAS 2 KRAS NCT A Phase Ib Study of MEK162 Plus BYL719 in Adult Patients With Selected Advanced Solid Tumors 3 KRAS NCT Trametinib and Navitoclax in Treating Patients With Advanced or Metastatic Solid Tumors 4 KRAS NCT Selumetinib and Cyclosporine in Treating Patients With Advanced Solid Tumors or Advanced or Metastatic Colorectal Cancer 5 KRAS, AKT1, TP53 NCT NCI-MPACT: Molecular Profiling-Based Assignment of Cancer Therapy for Patients With Advanced Solid Tumors Phase 1 MEK Phase 1/Phase 2 Phase 1/Phase 2 Phase 1 MEK Overall contact: Reference Study ID Number: GO _worldwide.htm, global.rochegenentechtrials@roche. com, (U.S. Only) CA (1), CO (1), CT (1), TN (1), TX (1) MEK, PI3K Overall contact: Array BioPharma Clinical Trial Call Center, CA (1), FL (1), IL (1), MA (1), NY (2), UT (1) MEK, Bcl- 2, BCL2L2 Phase 2 MTOR, PARP, Wee1, MEK *Note The trials listed above were matched to the biomarkers identified in this tumor; the list may not be comprehensive. Dana-Farber Cancer Institute: Massachusetts, USA, Geoffrey I. Shapiro, geoffrey_shapiro@dfci.harvard.edu, (MA) Massachusetts General Hospital Cancer Center: Massachusetts, USA, Ryan B. Corcoran, rbcorcoran@partners.org, (MA) CO (1), MO (2), NC (1), NJ (1), OH (1), PA (1), TX (1) Overall contact: Jennifer H Zlott, zlottjh@mail.nih.gov, (301) National Institutes of Health Clinical Center, 9000 Rockville Pike: Maryland, USA, For more information at the NIH Clinical Center contact National Cancer Institute Referral Office, (MD) 13

15 LBx Liquid Biopsy Test Report Patient ID: CM_01 Report ID: CellMax Life 1 Report Date: Dec 15, 2015 Client ID: POC SNOMED Concept Id: Disease: Carcinoma of colon (disorder) 3.2 Details on Biomarkers Detected 14

16 3.2 APC BIOMARKER RESULTS SUMMARY Marker Result Summary APC - MUTN (seq): E1536* APC-E1536* is an inactivating mutation. Inactivation of Apc results in the dysregulation of Wnt signaling through beta-catenin. Inhibitors of the Wnt pathway may be relevant for patients with APC defects (Fu et al., 2011; ). There are currently no approved drugs targeted to APC defects or Wnt upregulation in solid tumors; however, several potential approaches, including Wnt pathway inhibitors, Cox-2 inhibitors, and TRAIL agonists, are in clinical trials (Zhang et al., 2010; , Tuynman et al., 2008; , Lu et al., 2009; ) BIOLOGICAL RELEVANCE of APC APC alterations in Colorectal carcinoma Molecular function Incidence in disease The mutation reported here is expected to truncate the Apc protein within the beta-catenin binding domain, and is therefore likely to result in a disruption of the ability of the Apc protein to bind to beta-catenin, which in turn may upregulate Wnt signaling (Dikovskaya et al., 2010; , Eklof et al., 2001; , Liu et al., 2006; ). APC mutations have been found in 49% (1071/2176) of colorectal carcinoma samples analyzed in COSMIC (Sep 2015), and in 64% (165/259) of sequenced tumors in the Colorectal Adenocarcinoma TCGA dataset (cbioportal for Cancer Genomics, Sep 2015). Scientific studies have reported somatic mutations in the APC gene in 17-56% of colorectal cancer samples, as well as one study reporting germline mutations of APC in 37% (586/1591) of cases of familial adenomatous polyposis (Chen et al., 2013; , Yu et al., 2015; , Su et al., 2014; , Stachler et al., 2015; , Kerr et al., 2013; ). 15

17 3.2.3 CLINICAL RELEVANCE of APC APC alterations in Colorectal carcinoma Role in disease Effect on drug sensitivity APC is a tumor suppressor gene that was originally characterized based on the prominent role that inactivation of Apc plays in colorectal carcinogenesis; however, APC mutation and Wnt/beta-catenin pathway activation have subsequently been implicated in other tumor types as well (Fu et al., 2011; , Giles et al., 2003; , Prosperi and Goss, 2010; ). In the absence of functional Apc, beta-catenin accumulates and is translocated to the nucleus, where it promotes the transcription of genes promoting cellular proliferation (Hisamuddin and Yang, 2006; ). In addition, Apc has been reported to play a role in microtubule spindle formation and chromosomal segregation (Kaplan et al., 2001; , Green and Kaplan, 2003; , Fodde et al., 2001; ). A preclinical study in a mouse model of mucinous colorectal adenocarcinoma reported that allelic loss of APC in combination with a dominant active PI3K resulted in increased tumor number and size, as well as more aggressive and less differentiated tumors as compared with mice expressing an activated PI3K alone (Deming et al., 2014; ). Another preclinical study using transgenic mouse models reported that shrna-mediated conditional loss of APC together with KRASactivating and/or TP53-null mutations led to the development of either tubular adenoma or intramucosal adenocarcinoma tumors in the colon; re-expression of APC in these models led to increased cellular differentiation and tumor regression (Dow et al., 2015; ). There are currently no approved therapies that target Apc deficiency in cancer; however, several potential therapies, including Wnt pathway inhibitors, are in clinical trials. Cox-2 inhibitors, such as celecoxib, as well as other agents, including quercetin, may reduce Wnt signaling (Tuynman et al., 2008; , Park et al., 2005; , Lu et al., 2009; ). In addition, preclinical studies have reported that Apc inactivation or beta-catenin activation confer synthetic lethality when TRAIL receptors are upregulated and the TRAIL death receptor program is activated (Zhang et al., 2010; ). TRAIL agonists are currently in clinical trials in some cancer types CLINICAL EVIDENCE in Colorectal carcinoma APC alterations in Colorectal carcinoma FDA Approved Phase III Data Phase II Data Phase I Data Preclinical None. None. A Phase 2 clinical trial of celecoxib with irinotecan and capecitabine in colorectal carcinoma patients reported objective response in 41% (21/51) of patients; the addition of celecoxib did not significantly improve the response rate compared with chemotherapy alone (El-Rayes et al., 2008; ). A meta-analysis of clinical trials evaluating the effectiveness of celecoxib in cancer patients has reported finding a significant overall response rate in colorectal cancer patients; celecoxib therapy was significantly associated with an increase of cardiovascular effects (Chen et al., 2014; ). A Phase 1 trial of PRI-724 in 18 patients with advanced solid tumors reported acceptable toxicity, and three patients with colon cancer reported stable disease for 8, 10, and 12 weeks (El-Khoueiry et al., 2013; ASCO 2013, Abstract 2501). Early results from a Phase 1 study of the Wnt pathway inhibitor OMP-54F28 (FZD8-Fc) in solid tumors has reported that OMP-54F28 was well tolerated through 10 mg/kg, and stable disease was achieved in 18% (3/17) of patients for 2-3 months (Smith et al., 2013; AACR 2013, Abstract B79). N/A: Preclinical data are not presented when higher level data are available. 16

18 3.2.5 SAMPLE RELEVANT THERAPIES Therapies targeting COX-2 Drug Trade Name Target/Rationale Current Status Aspirin Ecotrin Cox-1,2 inhibitor, nonsteroidal antiinflammatory. Phase 3 (Colorectal carcinoma) FDA Approved (Pain) Celecoxib Celebrex Cox-2 inhibitor, nonsteroidal antiinflammatory. FDA Approved (Rheumatoid arthritis, Phase 3 (Colorectal carcinoma) Osteoarthritis) Etoricoxib Arcoxia Cox-2 inhibitor, nonsteroidal antiinflammatory. Phase 2 (Melanoma, Prostate carcinoma) Apricoxib Cox-2 inhibitor, nonsteroidal antiinflammatory. Phase 2 (Pancreatic carcinoma, Non-small cell lung carcinoma (NSCLC), Breast carcinoma) Therapies targeting Porcupine Drug Trade Name Target/Rationale Current Status LGK974 Porcupine inhibitor, inhibits Wnt signaling. Phase 1 (Colorectal carcinoma) Phase 1 (Solid Tumor) ETC Porcupine inhibitor, inhibits Wnt signaling. Phase 1 (Solid Tumor) Therapies targeting wnt Drug Trade Name Target/Rationale Current Status OTSA101 anti-fzd10 monoclonal antibody, Wnt antagonist. Phase 1 (Solid Tumor) OMP-54F28 Fzd8 fusion protein, Wnt antagonist. Phase 1 (Solid Tumor) Therapies targeting beta-catenin Drug Trade Name Target/Rationale Current Status PRI-724 CBP/beta-catenin inhibitor. Phase 2 (Colorectal carcinoma) Phase 2 (Signet ring colon carcinoma, Acute myelocytic leukemia (AML), Chronic myelocytic leukemia (CML)) 17

19 3.2.6 BIOMARKER-MATCHED CLINICAL TRIALS Trials Prioritized By Clinical Specificity* Markers TrialID Title Phase Targets Locations/contact 1 APC NCT Oxaliplatin, Leucovorin Calcium, and Fluorouracil With or Without Celecoxib in Treating Patients With Stage III Colon Cancer Previously Treated With Surgery 2 APC NCT A Trial of Maintenance ADAPT Therapy With Capecitabine and Celecoxib in Patients With Metastatic Colorectal Cancer 3 APC NCT Chemokine-Modulatory Regimen for Recurrent Resectable Colorectal Cancer 4 APC NCT A Study of LGK974 in Patients With Malignancies Dependent on Wnt Ligands 5 APC NCT Capecitabine and Celecoxib in Patients With Solid Cancers That Have Been Previously Treated With Standard Therapies Phase 3 COX-2, TYMS Phase 2 COX-2 Phase 1/Phase 2 COX-2, TLR3 Phase 1 Porcupine *Note The trials listed above were matched to the biomarkers identified in this tumor; the list may not be comprehensive. Overall contact: Jeffrey A. Meyerhardt, MD, MPH, AK (6), AL (1), AR (1), CA (67), CO (37), CT (5), DC (4), DE (10), FL (6), GA (7), HI (10), IA (26), ID (7), IL (60), IN (17), KS (25), KY (13), LA (12), MA (14), MD (8), ME (5), MI (39), MN (29), MO (34), MS (3), MT (9), NC (30), ND (5), NE (16), NH (6), NJ (10), NM (4), NV (12), NY (17), OH (65), OK (4), OR (11), PA (44), RI (3), SC (15), SD (5), TN (7), TX (7), VA (9), VT (4), WA (49), WI (47), WV (2), WY (2) Fred Hutchinson Cancer Research Center/University of Washington Cancer Consortium: Washington, USA, Edward H. Lin, elin@seattlecca.org, (WA) Overall contact: Amer H Zureikat, MD, zureikatah@upmc.edu, UPMC Hillman Cancer Center: Pennsylvania, USA, (PA) Overall contact: Novartis Pharmaceuticals, MA (1), MD (1), MI (2), TX (1) N/A COX-2 University of Chicago Comprehensive Cancer Center: Illinois, USA, Manish R. Sharma, M.D., msharma@medicine.bsd.uchicago.e du, (IL) 18

20 Trials Prioritized By Region* Markers TrialID Title Phase Targets Locations/contact 1 APC NCT Oxaliplatin, Leucovorin Calcium, and Fluorouracil With or Without Celecoxib in Treating Patients With Stage III Colon Cancer Previously Treated With Surgery 2 APC NCT A Trial of Maintenance ADAPT Therapy With Capecitabine and Celecoxib in Patients With Metastatic Colorectal Cancer 3 APC NCT Chemokine-Modulatory Regimen for Recurrent Resectable Colorectal Cancer 4 APC NCT A Study of LGK974 in Patients With Malignancies Dependent on Wnt Ligands 5 APC NCT Capecitabine and Celecoxib in Patients With Solid Cancers That Have Been Previously Treated With Standard Therapies Phase 3 COX-2, TYMS Phase 2 COX-2 Phase 1/Phase 2 COX-2, TLR3 Phase 1 Porcupine *Note The trials listed above were matched to the biomarkers identified in this tumor; the list may not be comprehensive. Overall contact: Jeffrey A. Meyerhardt, MD, MPH, AK (6), AL (1), AR (1), CA (67), CO (37), CT (5), DC (4), DE (10), FL (6), GA (7), HI (10), IA (26), ID (7), IL (60), IN (17), KS (25), KY (13), LA (12), MA (14), MD (8), ME (5), MI (39), MN (29), MO (34), MS (3), MT (9), NC (30), ND (5), NE (16), NH (6), NJ (10), NM (4), NV (12), NY (17), OH (65), OK (4), OR (11), PA (44), RI (3), SC (15), SD (5), TN (7), TX (7), VA (9), VT (4), WA (49), WI (47), WV (2), WY (2) Fred Hutchinson Cancer Research Center/University of Washington Cancer Consortium: Washington, USA, Edward H. Lin, elin@seattlecca.org, (WA) Overall contact: Amer H Zureikat, MD, zureikatah@upmc.edu, UPMC Hillman Cancer Center: Pennsylvania, USA, (PA) Overall contact: Novartis Pharmaceuticals, MA (1), MD (1), MI (2), TX (1) N/A COX-2 University of Chicago Comprehensive Cancer Center: Illinois, USA, Manish R. Sharma, M.D., msharma@medicine.bsd.uchicago.e du, (IL) 19

21 LBx Liquid Biopsy Test Report Patient ID: CM_01 Report ID: CellMax Life 1 Report Date: Dec 15, 2015 Client ID: POC SNOMED Concept Id: Disease: Carcinoma of colon (disorder) 3.3 Details on Biomarkers Detected 20

22 3.3 PI3K/Akt/mTOR BIOMARKER RESULTS SUMMARY Marker Result Summary AKT1 - MUTN (seq): E17K AKT1-E17K is an activating mutation. Amplification or mutation of AKT1, which encodes the Akt1 protein, may lead to activation of Akt signaling and therefore may result in sensitivity to Akt pathway inhibitors. Inhibitors of Akt are currently under investigation in clinical trials (Jo et al., 2011; , Kim et al., 2010; ). Inhibitors of the downstream protein mtor, including everolimus and temsirolimus, have been approved in some tumor types and may also be relevant in the case of Akt activation (Baselga et al., 2012; , Motzer et al., 2008; , Hudes et al., 2007; ) BIOLOGICAL RELEVANCE of AKT1 AKT1 alterations in Colorectal carcinoma Molecular function Incidence in disease AKT1 E17K is a missense mutation within the Akt1 pleckstrin homology domain; E17K has been reported to result in constitutive Akt1 activation due to PI3K-independent recruitment to the cell membrane (Carpten et al., 2007; ). Preclinical studies have also shown that AKT1 E17K promotes cell growth, colony formation, and migration and invasion in cell lines, as well as tumor formation in mice (Beaver et al., 2013; ). AKT1 mutations have been reported in 1.4% (20/1413) of colorectal carcinoma samples analyzed in COSMIC (Aug 2015) and in less than 1% (2/223) of sequenced tumors in the Colorectal Adenocarcinoma TCGA dataset (cbioportal for Cancer Genomics, Aug 2015). A study reported AKT1 mutations in 1.3% (4/311) colorectal carcinoma samples analyzed (Stachler et al., 2015; ) CLINICAL RELEVANCE of AKT1 AKT1 alterations in Colorectal carcinoma Role in disease Effect on drug sensitivity Although Akt activation has been shown to be insufficient to induce tumor formation on its own, it has been shown to accelerate tumor formation and increase metastasis in animal models of cancer (Hutchinson et al., 2001; , Dillon et al., 2009; , Hutchinson et al., 2004; ). Overexpression of phosphorylated Akt (p-akt) is thought to occur early in colorectal carcinoma tumorigenesis and to play a role in tumor progression (Roy et al., 2002; , Itoh et al., 2002; , Johnson et al., 2010; , Henderson-Jackson et al., 2010; ). Preclinical studies suggest that Akt signaling may regulate epithelial-mesenchymal transition in colorectal cancer (Suman et al., 2013; ). Numerous targeted therapies are under investigation for the PI3K/Akt/mTOR pathway. AKT1 E17K mutants are not expected to be sensitive to inhibition of PI3K, because the aberration in the PH domain allows them to be recruited to the cell membrane independent of PI3K activity (Carpten et al., 2007; ). However, inhibitors of Akt itself, or downstream mtor inhibitors, may be effective. Drugs that block that catalytic activity of Akt are currently being examined in clinical trials. Additional small molecules that block the recruitment of Akt to the membrane are under preclinical investigation (Jo et al., 2011; , Kim et al., 2010; ). The mtor inhibitors everolimus and temsirolimus, which have been approved by the FDA in some tumor types, as well as other mtor inhibitors, are being tested in clinical trials, both alone and in combination with other therapies. Targeting mtorc1/2 is another treatment strategy that is currently under investigation, and several inhibitors targeting the mtorc1/raptor and mtorc2/rictor complexes are being tested in early phase clinical trials (Grunt and Mariani, 2013; ). 21

23 3.3.4 CLINICAL EVIDENCE in Colorectal carcinoma AKT1 alterations in Colorectal carcinoma FDA Approved Phase III Data Phase II Data Phase I Data Preclinical None. Although a Phase 2 study of perifosine, an Akt/PIK3 inhibitor, in combination with capecitabine in refractory metastatic colorectal cancer patients reported an overall response rate of 20% with the combination versus 7% for capecitabine alone, a Phase 3 study of this combination reported no benefit of perifosine plus capecitabine over capecitabine alone (Bendell et al., 2012; ASCO 2012, Abstract LBA3501, Bendell et al., 2011; ). A Phase 2 clinical trial of temsirolimus as a single agent or in combination with irinotecan in patients with KRAS-mutant colorectal cancer has reported stable disease in 38% (24/64) and 63% (22/35) of patients on monotherapy or combination therapy, respectively; however, all patients who exhibited tumor reduction were found to have low levels of mutated KRAS in plasma samples (Spindler et al., 2013; ). A Phase 2 trial of MK-2206 and selumetinib treatment in 21 colorectal cancer patients, including KRAS wild-type and KRAS mutant cases, has reported no objective responses. Furthermore, no tumors showed at least a 70% reduction of both perk and pakt expression levels after treatment (Do et al., 2015; ). A Phase 2 trial of everolimus in heavily pretreated colorectal cancer patients reported no significant efficacy; however, patients were not selected according to mutational or activity status of any PI3K/Akt/mTOR pathway members (Ng et al., 2013; ). In another Phase 2 study, combination of everolimus with bevacizumab in patients with refractory metastatic colorectal cancer demonstrated stable disease in 15/50 patients (30%) and several minor responses (16%). Median progression-free survival was 2.3 months, and 26% of patients demonstrated stable disease for greater than six months (Altomare et al., 2011; ). A multicenter Phase 2 study of everolimus combined with tivozanib in colorectal cancer has reported stable disease in 50% (20/40) of patients; the authors of the study suggest that this combination was well tolerated and displayed activity in refractory, metastatic colorectal cancer (Wolpin et al., 2013; ). A Phase 1 study of 66 advanced solid tumor patients treated with GSK reported a partial response in one anal cancer patient, and stable disease for at least six months in 2/12 endometrial cancer and 2/9 prostate cancer patients (patients with stable disease had PIK3CA mutation and/or PTEN loss); GSK was reportedly well tolerated (Burris et al., 2011; ASCO 2011, Abstract 3003). Studies of the Akt antisense oligonucleotide RX-0201 have reported efficacy in growth inhibition of cancer cell lines and xenograft models (Yoon et al., 2009; , Pal et al., 2010; ). 22

24 3.3.5 SAMPLE RELEVANT THERAPIES Therapies targeting AKT Drug Trade Name Target/Rationale Current Status Perifosine Akt inhibitor induces apoptosis; mechanism Phase 3 (Colorectal carcinoma) of action is context-specific. Phase 3 (Multiple myeloma) MK-2206 Akt inhibitor. Phase 2 (Colorectal carcinoma) Phase 2 (Gastric carcinoma, Neuroendocrine carcinoma, Adenocarcinoma of the nasopharynx, Glioblastoma, Gallbladder carcinoma, Hepatocellular carcinoma, and various other cancers) AZD5363 Akt inhibitor. Phase 1 (Solid Tumor) Phase 2 (Gastric carcinoma, Non-small cell lung carcinoma (NSCLC), Prostate carcinoma, Endometrial carcinoma, Breast carcinoma) GSK Akt inhibitor. Phase 1 (Solid Tumor) Phase 2 (Melanoma, Endometrial carcinoma, Ovarian carcinoma, Breast carcinoma, Cervical carcinoma) GDC-0068 Akt inhibitor. Phase 1 (Colorectal carcinoma) Phase 2 (Prostate carcinoma) RX-0201 Archexin Akt antisense oligonucleotide. Phase 1 (Solid Tumor) Phase 2 (Pancreatic carcinoma, Renal cell carcinoma) Afuresertib Akt inhibitor. Phase 2 (Leukemia, Ovarian epithelial carcinoma, Chronic lymphocytic leukemia (CLL), Multiple myeloma) ARQ 092 Akt inhibitor. Phase 1 (Colorectal carcinoma) Phase 1 (Pancreatic carcinoma, Non-small cell lung carcinoma (NSCLC), Endometrial carcinoma, Ovarian carcinoma, Breast carcinoma) 23

25 Therapies targeting MTOR Drug Trade Name Target/Rationale Current Status Everolimus Afinitor mtor kinase inhibitor, immunosuppressant. Phase 2 (Colorectal carcinoma) FDA Approved (Pancreatic neuroendocrine tumor, Renal cell carcinoma, Breast carcinoma, Subependymal giant cell astrocytoma) Temsirolimus Torisel mtor inhibitor. Phase 2 (Colorectal carcinoma) FDA Approved (Renal cell carcinoma) PF PI3K/mTOR inhibitor. Phase 2 (Colorectal carcinoma) Phase 2 (Endometrial carcinoma) LY PI3K/mTOR dual inhibitor. Phase 1 (Solid Tumor) Phase 2 (Non-small cell lung carcinoma (NSCLC), Prostate carcinoma) VS-5584 PI3K/mTOR kinase inhibitor. Phase 1 (Solid Tumor) Therapies targeting mtorc1 Drug Trade Name Target/Rationale Current Status CC-223 Dual mtorc1/mtorc2 inhibitor. Phase 1 (Solid Tumor) Phase 2 (Hepatocellular carcinoma, Bladder neuroendocrine carcinoma, Gastrointestinal neuroendocrine carcinoma, Diffuse large B- cell lymphoma) INK128 Dual mtorc1/mtorc2 inhibitor. Phase 1 (Solid Tumor) Phase 2 (Non-small cell lung carcinoma (NSCLC), Prostate carcinoma, Thyroid carcinoma, Breast carcinoma) CC-115 DNA-PK/mTOR protein complex (TORC1/2) dual kinase inhibitor. Phase 1 (Solid Tumor) 24

26 3.3.6 BIOMARKER-MATCHED CLINICAL TRIALS Trials Prioritized By Clinical Specificity* Markers TrialID Title Phase Targets Locations/contact 1 AKT1 NCT Safety, Tolerability & Potential Anti-cancer Activity of Increasing Doses of AZD5363 in Different Treatment Schedules 2 AKT1 NCT Phase I Dose Escalation Study With an Allosteric AKT 1/2 Inhibitor in Patients 3 AKT1 NCT Phase I Dose Escalation Study of VS-5584 in Subjects With Advanced Non-Hematologic Malignancies or Lymphoma 4 AKT1 NCT Phase 1 Dose Escalation Study of ARQ 092 in Adult Subjects With Advanced Solid Tumors and Recurrent Malignant Lymphoma 5 AKT1 NCT A Study of LY in Participants With Advanced Cancer Phase 1 AKT Phase 1 AKT2, AKT1 Phase 1 MTOR, PI3K Phase 1 AKT Phase 1 MTOR, PI3K, ER, TYMS *Note The trials listed above were matched to the biomarkers identified in this tumor; the list may not be comprehensive. Overall contact: AstraZeneca Clinical Study Information Center, information.center@astrazeneca.co m, CA (2), CO (1), CT (1), MA (1), NY (1), OK (1), OR (1), SC (1), TN (1), TX (1) Overall contact: Bayer Clinical Trials Contact, clinical-trialscontact@bayerhealthcare.com CA (1), MA (1), MO (1), TX (1) Overall contact: Marie Payton, mpayton@verastem.com, Scottsdale Healthcare: Arizona, USA, Joyce Schaffer, MSN, RN, AOCNS, joschaffer@shc.org, (AZ) Cedars-Sinai Medical Center: California, USA, Katherine Rosenthal, RN, BSN, OCN, CCRP, Katherine.Rosenthal@cshs.org, (CA) Memorial Sloan Kettering Cancer Center: New York, USA, Anna Varghese, MD, varghesea@mskcc.org, (NY) Tennessee Oncology: Tennessee, USA, asksarah, ASKSARAH@scresearch.net, (TN) Overall contact: ArQule, Inc., ClinicalTrials@arqule.com, AL (1), AZ (1), FL (1), GA (2), IN (1), TX (1) Overall contact: There may be multiple sites in this clinical trial CTLILLY ( ) or, NY (1), OK (1), PA (1), TN (2) 25

27 Trials Prioritized By Region* Markers TrialID Title Phase Targets Locations/contact 1 AKT1 NCT Safety, Tolerability & Potential Anti-cancer Activity of Increasing Doses of AZD5363 in Different Treatment Schedules 2 AKT1 NCT Phase I Dose Escalation Study With an Allosteric AKT 1/2 Inhibitor in Patients 3 AKT1 NCT Phase I Dose Escalation Study of VS-5584 in Subjects With Advanced Non-Hematologic Malignancies or Lymphoma 4 AKT1 NCT Phase 1 Dose Escalation Study of ARQ 092 in Adult Subjects With Advanced Solid Tumors and Recurrent Malignant Lymphoma 5 AKT1 NCT A Study of LY in Participants With Advanced Cancer Phase 1 AKT Phase 1 AKT2, AKT1 Phase 1 MTOR, PI3K Phase 1 AKT Phase 1 MTOR, PI3K, ER, TYMS *Note The trials listed above were matched to the biomarkers identified in this tumor; the list may not be comprehensive. Overall contact: AstraZeneca Clinical Study Information Center, information.center@astrazeneca.co m, CA (2), CO (1), CT (1), MA (1), NY (1), OK (1), OR (1), SC (1), TN (1), TX (1) Overall contact: Bayer Clinical Trials Contact, clinical-trialscontact@bayerhealthcare.com CA (1), MA (1), MO (1), TX (1) Overall contact: Marie Payton, mpayton@verastem.com, Scottsdale Healthcare: Arizona, USA, Joyce Schaffer, MSN, RN, AOCNS, joschaffer@shc.org, (AZ) Cedars-Sinai Medical Center: California, USA, Katherine Rosenthal, RN, BSN, OCN, CCRP, Katherine.Rosenthal@cshs.org, (CA) Memorial Sloan Kettering Cancer Center: New York, USA, Anna Varghese, MD, varghesea@mskcc.org, (NY) Tennessee Oncology: Tennessee, USA, asksarah, ASKSARAH@scresearch.net, (TN) Overall contact: ArQule, Inc., ClinicalTrials@arqule.com, AL (1), AZ (1), FL (1), GA (2), IN (1), TX (1) Overall contact: There may be multiple sites in this clinical trial CTLILLY ( ) or, NY (1), OK (1), PA (1), TN (2) 26

28 LBx Liquid Biopsy Test Report Patient ID: CM_01 Report ID: CellMax Life 1 Report Date: Dec 15, 2015 Client ID: POC SNOMED Concept Id: Disease: Carcinoma of colon (disorder) 3.4 Details on Biomarkers Detected 27

29 3.4 p BIOMARKER RESULTS SUMMARY Marker Result Summary TP53 - MUTN (seq): R175H TP53 is a tumor suppressor; alterations in TP53 may result in a loss of p53 function, yet an increase in the expression and stability of the mutant p53 protein in the nucleus, sometimes leading to oncogenic effects, including genomic instability and excessive cell proliferation (Levine, 1997; , Wang et al., 2005; , Koga et al., 2001; , Kato et al., 2003; , Houben et al., 2011; , Olivier et al., 2009; ). At present, there are no approved therapies targeting TP53 alterations, despite their high prevalence in cancer. Therapeutic approaches under investigation include gene therapy for TP53 and (dendritic cell-based) TP53 vaccines (Schuler et al., 2014; , Vermeij et al., 2011; , Saito et al., 2014; ). Tumors with TP53 mutations may be sensitive to the Wee1 inhibitor MK-1775, and clinical trials are currently underway for patients with solid tumors and hematologic malignancies (Hirai et al., 2010; , Bridges et al., 2011; ). Additional p53-targeted approaches under clinical investigation, which may be relevant in the context of certain TP53 alterations, include kevetrin and ALT-801 (Kumar et al., 2012; AACR 2012, Abstract 2874, Fishman et al., 2011; ). Aurora kinase A inhibitors are another therapeutic approach under investigation for TP53-mutated cancers (Vilgelm et al., 2015; , Li et al., 2015; , Katayama and Sen, 2011; , Tentler et al., 2015; , Kalous et al., 2013; ). However, as the alteration reported here has been shown to have oncogenic effects, these therapeutic approaches are not expected to be relevant. Some studies suggest that Hsp90 inhibitors may be effective in tumors with oncogenic TP53 alterations (Alexandrova et al., 2015; , Lin et al., 2008; ) BIOLOGICAL RELEVANCE of TP53 TP53 alterations in Colorectal carcinoma Molecular function Incidence in disease TP53 R175H is a missense alteration located within the DNA-binding domain (DBD) of the p53 protein (Joerger and Fersht, 2008; ). DBD mutations are thought to result in loss of function via the loss of transactivation of p53-dependent genes (Kato et al., 2003; ). TP53 R175H has been reported to result in substantially reduced transactivation capacity, as compared with wild-type TP53, in yeast assays (IARC TP53 Database, release R17) (Kato et al., 2003; , Petitjean et al., 2007; ). The R175H mutation has been predicted to play a role in maintaining the structure of the DNA-binding region, and has been shown to result in the nuclear accumulation of p53 protein and have a gain-of-function oncogenic role; mice expressing R175H develop tumors similar to that of mice lacking TP53 (Song et al., 2007; , Liu et al., 2010; , Sigal and Rotter, 2000; , Grugan et al., 2013; , Tsang et al., 2005; , Gurtner et al., 2010; , Cho et al., 1994; , Dong et al., 2009; ). TP53 mutations have been reported in 52% (2753/5316) of colorectal carcinoma samples analyzed in COSMIC (Oct 2015) and in 53% (119/223) of sequenced tumors in the Colorectal Adenocarcinoma TCGA dataset (cbioportal for Cancer Genomics, Oct 2015). Scientific studies in the literature have reported TP53 mutations in 20-60% of colorectal cancer cases (Goh et al., 1995; , Lin et al., 2014; , Peeters et al., 2013; , Mouradov et al., 2013; , Russo et al., 2014; , Chang et al., 2015; ). Studies have reported that TP53 mutations are more frequent in colorectal cancer patients of less than approximately 56 years old than in older age groups (Berg et al., 2010; , Russo et al., 2014; ). 28

30 3.4.3 CLINICAL RELEVANCE of TP53 TP53 alterations in Colorectal carcinoma Role in disease Effect on drug sensitivity Effect on drug resistance Loss of tumor suppressor p53, which is encoded by the TP53 gene, is common in aggressive advanced cancers (Brown et al., 2009; ). Carriers of a germline mutation in TP53 have Li-Fraumeni Syndrome, an inherited cancer syndrome resulting in multiple tumors in early adulthood, including breast cancer, brain tumors, and leukemias (Malkin et al., 1990; , Srivastava et al., 1991; , Santibáñez-Koref et al., 1991; ). Expression of p53 in normal cells is low; however, TP53 alterations, including those that result in loss of p53 tumor suppressor function, may lead to stabilization and increased expression of p53, particularly in the nucleus, and several studies have shown that it may have oncogenic gain-of-function effects (Wang et al., 2005; , Koga et al., 2001; , Kato et al., 2003; , Houben et al., 2011; , Olivier et al., 2009; ). At present, there are no approved therapies targeting TP53 alterations, despite their high prevalence in cancer. Therapeutic approaches under investigation include gene therapy for TP53 and (dendritic cell-based) TP53 vaccines (Schuler et al., 2014; , Vermeij et al., 2011; , Saito et al., 2014; ). Several other therapeutic strategies are currently in development for inactivating TP53 mutations (Bridges et al., 2011; , Kumar et al., 2012; AACR 2012, Abstract 2874, Fishman et al., 2011; , Li et al., 2015; ). However, as the alteration reported here has been shown to have oncogenic effects, these therapeutic approaches are not expected to be relevant. Some studies suggest that Hsp90 inhibitors may be effective in tumors with oncogenic TP53 alterations (Alexandrova et al., 2015; , Lin et al., 2008; ). One study of 68 metastatic colon cancer patients with known TP53 status reported no differences in response to oxaliplatin- or irinotecan-based chemotherapy between patients harboring a TP53-mutation and patients with wild-type TP53 (Netter et al., 2015; ). Mutations in TP53 may increase resistance to ionizing radiation therapy (El-Deiry, 2003; , Miyasaka et al., 2015; ). One study reported that oncogenic TP53 mutations were associated with increased platinum resistance in high grade serous ovarian cancer as compared with non-oncogenic TP53 mutations (Brachova et al., 2015; ) CLINICAL EVIDENCE in Colorectal carcinoma TP53 alterations in Colorectal carcinoma FDA Approved Phase III Data Phase II Data Phase I Data Preclinical None. None. A Phase 2 trial with 17 metastatic colorectal carcinoma patients treated with ganetespib reported stable disease greater than four months in two patients (12%) with durations of 6.8 and 5.1 months (Cercek et al., 2014; ). None. In preclinical studies in colorectal cancer cell lines, ganetespib increased cell death and enhanced sensitivity to radiation; ganetespib also increased the effects of capecitabine in a xenograft model of colorectal cancer (He et al., 2014; ). Inhibition of Hsp90 with ganetespib was also reported to lead to reduced EMT, invasion, and migration in colorectal cancer cells, and increased sensitivity to oxaliplatin and 5-fluorouracil in cell and animal models (Nagaraju et al., 2014; , Nagaraju et al., 2014; ). 29