Patient Name: GarciaSanchez, Jose Luis Patient DOB / Sex: 04/15/1940 / M NeoTYPE™Accession / CaseNo: 3255981 Analysis / NTP20-032233 Discovery Profile

Client 1234 Patient Name: Patient, Sample Ordering Physician(s): Sample Doctor, M.D. Sample Client Patient DOB / Sex: 01/01/1940 / M Treating Physician(s): Sample Doctor, M.D. Address Specimen Type: Paraffin Tissue Accession / CaseNo: 9999999 / XXX99-999999 City, ST 99999 Body Site: R ght Nephrourectomy, Kidney Ureter Collection Date: 07/23/2020 Phone: (555) 555-5555 i Fax: (555) 555-5555 Specimen ID: X99-99 Received Date: 09/09/2020 01:30:00 PM PDT MRN: 9999999 Report Date: 09/23/2020 12:42:36 PM EST Reason for Referral: UROTHELIAL CANCER, MALIGNANT NEOPLASM OF URINARY ORGAN, UNSPECIFIED

Results Summary

4 Clinically Significant PIK3CA E542K; RB1 S634*; TERT promoter c.-124C>T; TP53 E271K Variants Detected§ 2 Alterations Detected MYC Amplification: POSITIVE; PTEN Deletion: POSITIVE by FISH Immuno-Oncology Microsatellite Instability: MSI - Stable (MSS); PD-L1 22C3: EXPRESSED; Biomarkers Tumor Mutation Burden: HIGH

Additional Studies Pan-TRK: Not Expressed

NO abnormalities detected in the following genes: BRAF, BRCA1, BRCA2, EGFR, Pertinent Negatives KIT, KRAS, NRAS, PDGFRA Interpretation - High levels of MYC expression have been linked to aggressive cancer and poor prognosis. - Loss of PTEN may be associated with resistance to EGFR mAb inhibitors, but may respond to PI3K/AKT/mTOR and PARP inhibitors. - The expression of PD-L1 suggests response to immunotherapy with anti-PD-1 or anti-PD-L1, which are FDA-approved for diverse solid tumor types. - High TMB independently predicts responsiveness to anti-PD-1 or anti-PD-L1 therapies which are FDA-approved for diverse solid tumor types (High TMB on this assay correlates with a value of at least 10 mutations/Mb as assessed by an FDA-approved companion diagnostic). § See full list of genes tested in Biomarkers Evaluated section at end of report. See Profile Results Detail for Variants of Unknown Clinical Significance.

Therapeutic Implications (Biomarkers Analyzed by FISH and IHC Excluded) Biomarker Tier Therapies approved Therapies approved Possible Therapy Potential Clinical in this indication in other indication Resistance (LoE°) Trials (LoE°) (LoE°) PIK3CAE542K 2 None Alpelisib (C), Copanlisib None Yes, See Clinical Trials (C) Section TERTpromoter c.-12 2 None None None None 4C>T RB1S634* 2 None None None None TP53E271K 2 None None None Yes, See Clinical Trials Section °Level of Evidence SAMPLE(LoE) – See level of evidence table for additional information

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Profile Results Detail

Molecular Testing Detail

Gene name Variant Amino Acid Nucleotide Consequence Mutant Allele Read Depth Change Change Frequency (%)

NM_006218.4: PIK3CA E542K p.E542K Missense 35.9 2208 c.1624G>A

promoter c.-124C> promoter c.-124C> NM_198253.3: TERT Upstream gene 21.8 197 T T c.-124C>T

NM_000321.2: RB1 S634* p.S634* Stop gained 47.8 2727 c.1901C>G

NM_000546.5: TP53 E271K p.E271K Missense 22.6 2063 c.811G>A

Variants of Unknown Clinical Significance

DOT1L R282T NM_032482.3:c.845G>C

FAT1 P2743S NM_005245.4:c.8227C>T

GNA13 E83Q NM_006572.6:c.247G>C

JAK2 K1053N NM_004972.3:c.3159G>C

KMT2D R5179L NM_003482.3:c.15536G>T

MTOR I2250M NM_004958.4:c.6750C>G

NBN G206V NM_002485.4:c.617G>T

NSD1 S1210L NM_022455.4:c.3629C>T

PRKDC E203K NM_006904.7:c.607G>A

RANBP2 F3108L NM_006267.5:c.9324C>G

SMARCA4 F933L NM_001128844.2:c.2799C>G

SPTA1 R1477C NM_003126.4:c.4429C>T

TBX3 S55W NM_016569.4:c.164C>G

TSC1 H206Q NM_000368.5:c.618T>G

XPO1 L744F NM_003400.4:c.2232G>C

Biomarker/Assay Results Biomarker/Assay Results Microsatellite Instability MSI - Stable (MSS) Tumor Mutation Burden Result High 14.9 Mutations/Mb

FISH Testing Detail Test SAMPLEResults ISCN Data ALK FISH Negative nuc ish(ALKx1~>1)[49] BRAF FISH Not Detected (Atypical) nuc ish(BRAFx3~>3)[27/50]

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Test Results ISCN Data

HER2 Non-Breast FISH Negative nuc ish(CEN17x1.9,HER2x1.9)[50] Average HER2 Signals 1.9 Average CEN17 Signals 1.9 HER2/CEN17 Ratio 1 MET FISH Negative nuc ish(CEN7x2.8,METx1.9)[50] MYC FISH Positive nuc ish(MYC amp)[27/50] PDGFRa Amplification by FISH Not Detected (Atypical) nuc ish(CEN4x2.4,PDGFRax2.4)[50] nuc ish(CEN10x2~>2,PTENx1)[17/50]/(CEN10x3~>3,PTENx2)[15/50]/ PTEN FISH Positive (CEN10x2,PTENx0)[6/50] RET FISH Not Detected (Atypical) nuc ish(RETx1)[35/50] ROS1 FISH Negative nuc ish(ROS1x1~>1)[46]

BRAF MYC (8q24) PDGFRa amp PTEN (10q23)

XXX00-000000 XXX00-000000 XXX00-000000 XXX00-000000 XXX00-000000 XXX00-000000 XXX00-000000 XXX00-000000 Patient Sample BRAF ALL.jpg Patient Sample MYC ALL.jpg Patient Sample PDGFRa Patient Sample PTEN ALL.jpg ALL.jpg

RET

XXX00-000000 XXX00-000000 Patient Sample RET ALL.jpg

Histology Testing Detail Test Result Pan-TRK Not Expressed PD-L1 22C3 FDA (KEYTRUDA®)SAMPLE for Gastric/GEA CPS >/=1 (PD-L1 Expression) Combined Positive Score 70

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PD-L1 22C3 FDA (KEYTRUDA®) for Pan-TRK Gastric/GEA

XXX11-111111 XXX11-111111

Detected Alterations

PIK3CA E542K - Biomarker Information Clinical PIK3CA-E542K is an activating mutation. PIK3CA encodes the protein p110-alpha, which is the catalytic subunit of Relevance phosphatidylinositol 3-kinase (PI3K). The PI3K pathway is involved in cell signaling that regulates a number of critical cellular functions, including cell growth, proliferation, differentiation, motility, and survival (29, 30). Activating PIK3CA alterations may predict sensitivity to PI3K/Akt/mTOR pathway inhibitors, several of which are currently being tested in clinical trials (31, 32). In addition, the p110-alpha inhibitor alpelisib has been approved by the FDA for the treatment of postmenopausal women, and men, with PIK3CA-mutated, hormone receptor-positive, Her2-negative advanced or metastatic breast cancer who experience disease progression on or following an endocrine-based therapy (33). PIK3CA E542K has been reported in an ER-positive breast cancer case who developed fulvestrant resistance; E542K was reported to decrease fulvestrant sensitivity in a breast cancer cell model (34). Drug Activating PIK3CA alterations may predict sensitivity to PI3K/Akt/mTOR pathway inhibitors, several of which are Sensitivity currently being tested in clinical trials (31, 32). While PIK3CA activating alterations have been suggested to predict sensitivity to the mTOR inhibitors everolimus and temsirolimus, results from clinical studies have been mixed, with several reporting no associations between PIK3CA mutational status and response to therapy (31, 35, 36, 37, 38). Other agents that target mTOR, including dual mTORC1/mTORC2 inhibitors and dual PI3K/mTOR inhibitors, are currently in preclinical and clinical development (39, 40, 41). In addition, the p110-alpha inhibitor alpelisib has been approved by the FDA for the treatment of postmenopausal women, and men, with PIK3CA-mutated, hormone receptor-positive, Her2- negative advanced or metastatic breast cancer who experience disease progression on or following an endocrine-based therapy (33). Possible PIK3CA E542K has been reported in a fulvestrant-resistant ER-positive breast cancer case; E542K was reported to Therapy decrease fulvestrant sensitivity in a breast cancer cell model (34). Resistance Molecular PIK3CA E542K is a missense alteration occurring in the helical domain of the p110-alpha protein, at a mutational Function hotspot; this alteration has been shown to drive increased lipid kinase activity and be an oncogenic mutation with transforming activity (34, 42, 43). PIK3CA E542K has been reported in an ER-positive breast cancer case who developed fulvestrant resistance; E542K was reported to decrease fulvestrant sensitivity in a breast cancer cell model (34). Role in PIK3CA mutations are not mutually exclusive with EGFR or KRAS or BRAF mutations, and are associated with Disease increased PI3K signaling and increased activation of Akt (44, 45). PIK3CA mutations have been reported to be early events in urothelial carcinogenesis and are reported to be mutually exclusive with AKT1, but associated with FGFR3 and KRAS mutations; in addition, activation of the PI3K/Akt pathway has been reported to be common across different stages of bladder urothelial carcinoma (46, 47, 48, 49). PIK3CA mutations has been significantly associated with low- grade non-invasive papillary carcinoma as compared with high-grade non-invasive papillary carcinoma and carcinoma in situ in a study of 373 bladder urothelial carcinoma cases (50). Prevalence PIK3CA mutations have been reported in 18% (4/22) of Renal pelvis urothelial carcinoma samples analyzed in COSMIC (MaySAMPLE 2020). In the literature, PIK3CA mutations have been reported in approximately 5-27% of bladder urothelial carcinoma specimens (51, 52, 50, 49, 46, 53, 54). In addition, PIK3CA mutations have been reported in 10-26% of upper tract urothelial carcinoma samples (55, 56, 51).

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PIK3CA E542K - Clinical Evidence from Completed Clinical Trials Phase 2 Preliminary results from a Phase 2 study of buparlisib in 13 evaluable metastatic urothelial carcinoma patients reported a median progression-free survival of 2.8 months, with one partial response and six patients with stable disease as per RECIST criteria (57). Phase 1 A Phase 1 study of alpelisib in PIK3CA-altered advanced solid tumors has reported an overall response rate of 6% (8/134), including one complete and seven partial responses, as well as stable disease in 52% (70/134) of patients. The safety profile was also reported to be tolerable (58). A Phase 1 study of alpelisib monotherapy in 33 Japanese patients with advanced solid tumors has reported overall response rate, disease control rate, and median progression-free survival at 350 mg/day of 3%, 57.6%, and 3.4 months, respectively. The most common treatment-related adverse events were hyperglycemia, maculopapular rash, and diarrhea observed in 48.5%, 48.5%, and 45.5%, of patients, respectively (59). A Phase 1 study of copanlisib in 48 patients with advanced solid tumors reported one complete response in an endometrial carcinoma patient with PIK3CA activation and PTEN loss, as well as two partial responses in metastatic breast cancer patients (60). A Phase 1 trial of capivasertib (AZD5363) in 90 solid tumor patients has reported stable disease for more than 6 and 12 weeks in 30% (27/90) and 7% (6/90) of patients, respectively, and one partial response in a cervical cancer patient with a PIK3CA mutation. In an expansion cohort of patients with PIK3CA mutations, confirmed RECIST responses were observed in 1/28 and 8% (2/26) of breast and gynecologic cancer patients, respectively, resulting in termination of further enrollment (61). A Phase 1 study of capivasertib (AZD5363) in 41 Japanese solid tumor patients has reported confirmed partial responses in 5% (2/37) of evaluable patients, both with the AKT1 E17K mutation, and stable disease in 27% (10/37) of patients. Grade 3 or higher treatment-related adverse events were observed in 58.5% of patients (62). A Phase 1 study of pan-Akt inhibitor MK-2206 in 33 patients with advanced solid tumors resulted in decreased Akt phosphorylation, and one patient with a pancreatic adenocarcinoma with PTEN loss and KRAS G12D mutation achieved 23% tumor shrinkage, and two patients with pancreatic neuroendocrine tumors had minor responses (63). A Phase 1 study of the Akt inhibitor MK-2206 combined with carboplatin/paclitaxel, docetaxel, or erlotinib in 60 evaluable advanced solid tumor patients reported a complete response in one patient with head and neck squamous cell carcinoma (HNSCC), and partial responses in patients with HNSCC, melanoma, endometrial cancer, neuroendocrine prostate cancer, non-small cell lung cancer, and cervical cancer; six patients also exhibited stable disease for at least six months (64). A Phase 1 study of MK-2206 combined with hydroxychloroquine in 35 patients with advanced solid tumors has reported best overall response of stable disease in 15% (5/34) of evaluable patients. Treatment related adverse events were reported in 94% (33/35) of patients, specifically adverse events attributed to MK-2206 and hydroxychloroquine were reported in 94% (33/35) and 37% (13/35) of patients, respectively (65).

TERT promoter c.-124C>T - Biomarker Information Clinical TERT-promoter -124C>T is an activating mutation. TERT encodes the telomerase reverse transcriptase (hTERT) Relevance protein and is frequently amplified in several cancer types (86, 87, 88). However, hypomorphic germline TERT mutations may cause dyskeratosis congenita, a cancer predisposition syndrome (89, 90, 91, 92). Several therapies targeting the RNA or protein components of telomerase, as well as the telomeres themselves, are in development; however, these therapies may have significant negative consequences for normal tissues as well (93, 94). Therefore, therapeutic strategies targeting TERT alterations must be carefully considered. Drug Therapies targeting telomeres or telomerase components have been in development, although discoveries regarding Sensitivity alternative roles in normal cells, as well as consequences of shortened telomeres, may limit their use (93, 94, 95, 96, 97). Molecular TERT promoter -124C>T, also known as C228T, chr5:1,295,228 C>T, or c.-124G>A, occurs prior to the transcriptional Function start site within the promoter of the TERT gene (98, 99). This alteration has been reported to result in increased transcriptional activity of the TERT promoter, hTERT protein expression, telomerase activity, and telomere length, as compared with wild-type TERT (100, 99, 101, 102). The TERT promoter -124C>T mutation has also been shown to drive tumor formation in a xenograft model using bladder cancer stem cells, while removal of this mutation decreased tumor formation (102). Role in TERT promoter mutations have been suggested to act as driver events in the malignant transformation of cells to Disease melanoma and perhaps other types of cancer (99). TERT promoter mutations have been significantly associated with distant metastasis in a study of 220 upper tract urothelial carcinoma cases, and have been suggested to be an early event in bladder urothelial carcinoma pathogenesis (103, 104, 105, 106, 107). Methylation of TERT has been significantly associated with tumor grade in a study of 108 urothelial carcinoma of the bladder patients and 105 normal cases (108). In addition, increased hTERT or TERT mRNA expression has been associated with increased invasion, as well as disease stage and grade in urothelial carcinoma (109, 110). Prevalence TERT mutations have been reported in 14% (1/7) of Renal pelvis urothelial carcinoma samples analyzed in COSMIC (MaySAMPLE 2020). TERT promoter mutation has been reported in 48-79% of urothelial carcinoma specimens analyzed in several studies in the literature (111, 112, 113, 114, 115, 107, 116).

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RB1 S634* - Biomarker Information Clinical RB1-S634* is an inactivating mutation. RB1 encodes the retinoblastoma protein (Rb), a tumor suppressor and Relevance negative regulator of the cell cycle. Loss of Rb function, which has been identified in many cancer types due to various mechanisms, results in the upregulation of transcription and cell proliferation (192, 193). Loss of RB1 has been associated with lack of response to Cdk4/6 inhibitors (194, 195, 196, 197, 198, 199, 200). At this time, there are no therapeutic options to target the inactivation of Rb. Preclinical studies are actively investigating possible therapies to address Rb inactivation, exploring avenues such as Aurora kinase inhibitors, Bcl-2 family inhibitors, and Notch pathway activation (201, 202, 203, 204). Drug At this time, there are no therapeutic options to target the inactivation of Rb. Preclinical studies are actively investigating Sensitivity possible therapies to address Rb inactivation, exploring avenues such as Aurora kinase inhibitors, Bcl-2 family inhibitors, and Notch pathway activation (205, 201, 202, 203). Loss of Rb function has been associated with increased sensitivity to cytotoxic agents in both preclinical studies and in patients with bladder or breast cancer (206, 193). The presence of RB1 alterations has been reported to predict pathologic response after neoadjuvant cisplatin-based chemotherapy in a prospective study of muscle-invasive bladder cancer cases (207). Possible The effect of Rb expression on chemoresistance is complex, as both Rb protein expression and loss of Rb protein have Therapy been associated with resistance to chemotherapeutics (208, 209, 210, 206, 211, 212). Loss of RB1 has been associated Resistance with lack of response to Cdk4/6 inhibitors (194, 195, 196, 197, 198, 199, 200). Molecular The alteration reported here is expected to truncate the 928 amino acid Rb protein and result in the loss of all or a Function portion of the A/B pocket, LIMD1 interaction, and/or C domains, as well as a probable nuclear localization signal (UniProt) (213). The Rb pocket domain, which consists of domain A and domain B, has been reported to be involved in the interaction of Rb with several proteins (213, 214, 215). In addition, amino acids 785-841 of the Rb protein have been reported to be required for growth suppression and E2F binding (216, 217). Due to the expected loss of multiple functional domains, this alteration is predicted to be inactivating. Role in RB1 inactivation has been shown to cause epigenetic deregulation of genes involved in several cancer pathways and is Disease thus speculated to play a key role in cancer development (218). Retinoblastoma, a malignant tumor of the retina, arises from mutations in both RB1 alleles. Hereditary retinoblastoma patients carry one RB1 germline mutation, which also increases their risk of developing a second type of cancer later in life (219). Studies in both bladder cancer and bladder urothelial carcinoma specifically have correlated decreased or a loss of Rb protein expression with higher tumor stage and muscle-invasive disease (220, 221, 222, 223, 224, 225). In addition, preclinical studies have shown that Rb loss in combination with loss of p53 expression induced urothelial carcinogenesis, although loss of either protein alone was insufficient for transformation (226, 227, 228). Prevalence Renal pelvis urothelial carcinoma samples have not been analyzed in COSMIC for mutations in RB1. RB1 mutations have been reported in 3.3% (3/91) of Upper urinary tract urothelial carcinoma samples analyzed in COSMIC (May 2020). In the scientific literature, RB1 mutations have been reported in 4-20% of bladder urothelial carcinoma samples (229, 179, 230, 231, 55, 232). Additional studies have reported RB1 mutations in 0-3% of upper tract urothelial carcinoma samples (229, 55, 179).

RB1 S634* - Clinical Evidence from Completed Clinical Trials Pre-Clinical A preclinical study has reported that treatment of bladder cancer cell lines with palbociclib resulted in decreased proliferation of Rb- positive cells, decreased Rb phosphorylation, and reduced total Rb protein levels (233).

TP53 E271K - Biomarker Information Clinical TP53-E271K is an inactivating mutation. TP53 is a tumor suppressor that encodes the p53 protein; alterations in TP53 Relevance 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 (234, 235, 236, 237, 238, 239). 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 (240, 241, 242). Tumors with TP53 mutations may be sensitive to the Wee1 inhibitor adavosertib (MK-1775), and clinical trials are currently underway for patients with solid tumors and hematologic malignancies (243, 244). Aurora kinase A inhibitors are another therapeutic approach under investigation for TP53-mutated cancers (245, 246,SAMPLE 247, 248, 249).

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TP53 E271K - Biomarker Information Drug At present, there are no approved therapies targeting TP53 alterations, despite their high prevalence in cancer. Sensitivity Therapeutic approaches under investigation include gene therapy for TP53 and (dendritic cell-based) TP53 vaccines (240, 241, 242). Inhibition of components of the DNA damage checkpoint, including Checkpoint Kinase 1 (Chk1) and Wee1, has been reported to enhance the activity of DNA-damaging agents in preclinical cancer models with deficiency of p53 function (250, 243, 244). Clinical trials of the Wee1 inhibitor adavosertib (MK-1775) are currently underway for patients with solid tumors and hematologic malignancies. Studies have reported Aurora kinase A to be activated in cells harboring TP53 mutation, and Aurora kinase A and B inhibitors have been reported to activate wild-type p53 in cellular assays; thus, tumors retaining a wild-type TP53 allele may benefit from Aurora kinase inhibitors (245, 246, 247, 248, 251, 252). Possible Therapy Mutations in TP53 may increase resistance to ionizing radiation therapy (253, 254). Resistance Molecular TP53 E271K is a missense alteration located within the DNA-binding domain (DBD) of the p53 protein (255). DBD Function mutations are thought to result in loss of function via the loss of transactivation of p53-dependent genes (237). TP53 E271K has been reported to result in reduced transactivation capacity, as compared with wild-type TP53, in yeast assays (IARC TP53 Database, release R20) (237, 256). Role in Loss of tumor suppressor p53, which is encoded by the TP53 gene, is common in aggressive advanced cancers (257). Disease 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 (258, 259, 260). 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 (235, 236, 237, 238, 239). TP53 alterations, including TP53 mutations and p53 protein accumulation, have been associated with high tumor grade and stage, as well as invasion in bladder carcinoma (261, 262, 51, 54, 263, 264, 265, 266, 267). The high frequency of TP53 and FGFR3 mutations in urothelial carcinoma has led to the suggestion that these tumors develop through at least two molecular pathways, one related to FGFR3, typically in less invasive tumors, and one related to TP53, characterized by higher grade invasive tumors (268, 269, 270). Certain TP53 alterations have been associated with exposure to the carcinogen aristolochic acid in upper urinary tract urothelial carcinoma samples (271, 272). Prevalence TP53 mutations have been reported in 41% (38/93) of Renal pelvis urothelial carcinoma samples analyzed in COSMIC (May 2020). TP53 mutations have been reported in 26-59% of urothelial carcinoma samples analyzed (262, 51, 273, 274).

TP53 E271K - Clinical Evidence from Completed Clinical Trials Phase 2 A Phase 2 study of alisertib in 20 evaluable advanced urothelial carcinoma patients reported partial responses in two patients and stable disease in seven patients, with a progression-free survival (PFS) rate of 13.6% at 6 months; two treatment-related deaths were reported (275). Phase 1 In a Phase 1 study of ENMD-2076 in 67 patients with advanced cancer, partial responses were seen in two patients with platinum- refractory ovarian carcinoma. Therapy was reported to be well-tolerated overall, with hypertension, nausea/vomiting, and fatigue being the most common adverse events (276). A Phase 1 trial of adavosertib (AZD1775, MK-1775) in 21 evaluable patients with refractory solid tumors, including seven patients with documented BRCA1/2 mutations, reported confirmed partial responses in one head and neck cancer and one ovarian cancer patient, both harboring BRCA1 mutations; however, no responses were seen in any of five patients with confirmed TP53 mutations (277). A Phase 1 trial of adavosertib alone or in combination with chemotherapy in patients with refractory solid tumors has reported confirmed or unconfirmed partial responses in 10% (17/176) of patients overall, including in patients with ovarian cancer, melanoma, breast cancer, head and neck cancer, colorectal cancer, and cutaneous squamous cell carcinoma. In patients with archived tumor tissue evaluable for sequence analysis, partial responses were reported in 21% (4/19) and 12% (4/33) of TP53 mutant and TP53 wild-type cases, respectively. Stable disease was reported in 53% (94/176) of patients overall (278). A Phase 1 study of adavosertib (AZD1775) in combination with irinotecan in 27 evaluable pediatric patients with relapsed/refractory solid tumors or central nervous system tumors has reported a confirmed partial response in an Ewing sarcoma patient and prolonged stable disease in two patients, one each with ependymoma and neuroblastoma. Grade 3 dehydration, which was experienced by two patients, was reported as a dose-limiting toxicity (279). A Phase 1 trial of SGT-53 in 11 patients with refractory cancer reported that the gene therapy complex was well SAMPLEtolerated with stable disease achieved in seven patients at six weeks and a median survival of 340 days; in addition, one tumor which was previously classified as inoperable was able to be resected (280). A Phase 1 trial of SGT-53 in combination with docetaxel in 14 patients with advanced cancer has reported three partial responses and two stable diseases per RECIST (281).

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Open & Recruiting Clinical Trials

Gene Trial ID Title Targets Phase Locations PIK3CA NCT03006172 To Evaluate the Safety, CDK4, CDK6, Phase 1 More than 5 locations. Please Tolerability, and ER, ESR1, PI3K, see clinical trial website Pharmacokinetics of GDC-0077 PRKAB1 for full list of trial sites: Single Agent in Participants With http://clinicaltrials.gov/show/ Solid Tumors and in Combination NCT03006172 With Endocrine and Targeted Therapies in Participants With Breast Cancer PIK3CA NCT04192981 GDC-0084 With Radiation MTOR, PI3K Phase 1 More than 5 locations. Please Therapy for People With see clinical trial website PIK3CA-Mutated Solid for full list of trial sites: Tumor Brain Metastases or http://clinicaltrials.gov/show/ Leptomeningeal Metastases NCT04192981 PIK3CA NCT03842228 Testing the Combination of the CD274, PARP, Phase 1 Houston, TX; Boston, MA Anti-cancer Drugs Copanlisib, PI3K Olaparib, and MEDI4736 (Durvalumab) in Patients With Advanced Solid Tumors With Selected Mutations PIK3CA NCT03065062 Study of the CDK4/6 Inhibitor CDK4, CDK6, Phase 1 Boston, MA Palbociclib (PD-0332991) in MTOR, PI3K Combination With the PI3K/ mTOR Inhibitor Gedatolisib (PF-05212384) for Patients With Advanced Squamous Cell Lung, Pancreatic, Head & Neck and Other Solid Tumors PIK3CA NCT04317105 Testing the Addition of an Anti- CTLA4, PDCD1, Phase 1/ Houston, TX cancer Drug, Copanlisib, to PI3K Phase 2 the Usual Immunotherapy (Nivolumab With or Without Ipilimumab) in Patients With Advanced Solid Cancers That Have Changes in the Following Genes: PIK3CA and PTEN TERT None None None None None RB1 None None None None None TP53 NCT04383938 Phase 1/2 Study of APR-246 PDCD1, TP53 Phase 1/ Saint Louis, MO; Houston, TX in Combination With Phase 2 Pembrolizumab in Subjects With Solid Tumor Malignancies TP53 NCT04555837 Alisertib and Pembrolizumab for AURKA, PDCD1 Phase 1/ Houston, TX the Treatment of Patients With Phase 2 Rb-deficient Head and Neck Squamous Cell Cancer TP53 NCT03654547 Safety of TT-00420 Monotherapy AURKA, AURKB Phase 1 Houston, TX in Patients With Advanced Solid Tumors and Triple Negative Breast Cancer TP53 NCT03968653 Study of Oral Debio 0123 in WEE1 Phase 1 Leiden, Netherlands; Nijmegen, Combination With Carboplatin in Netherlands; Groningen, SAMPLEParticipants With Advanced Solid Netherlands; Barcelona, Spain Tumors

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Gene Trial ID Title Targets Phase Locations TP53 NCT04462952 Study of Adavosertib(AZD1775) WEE1 Phase 1 Chuo-ku, Japan in Japanese Patients With Advanced Solid Tumours

NOTE: Although we make every attempt to ensure that the information provided is as accurate as possible, please note that the information provided in this report has been obtained through public domains that are updated constantly and should be researched by the physician or research professionals. NOT ALL TRIALS ARE INCLUDED. Please go to www.clinicaltrials.gov to perform a detailed search of available clinical trials as the trials provided in this report are not meant to be a complete list.

Test Description & Methodology

Test Description The NeoTYPE Discovery Profile analyzes 336 biomarkers through a combination of next-generation sequencing (NGS), FISH, and IHC as listed below. Test orders include summary interpretation of all results to help guide treatment decisions. If Pan-TRK IHC is expressed or equivocal, NTRK NGS Fusion Profile for NTRK1 fusions, NTRK2 fusions, and NTRK3 fusions will be added by reflex. Clinical Significance The NeoTYPE™ Discovery Profile for Solid Tumors is a test profile that combines 323 genes by NGS, 9 FISH, 1 IHC, and Tumor Mutation Burden to allow for the accurate and sensitive detection of genomic alterations in the genes most relevant to various solid tumor cancers. These genomic alterations include SNP’s, indels, rearrangements and other alterations. Testing can aid in the diagnosis of various diseases and provide information to develop strategies for the treatment and management of the underlying disease. The results obtained from the NeoTYPE™ Discovery Profile for Solid Tumors can also be used in current or future clinical research projects. Studies in lung cancer, melanoma and bladder cancers showed that objective response to checkpoint immunotherapy was predicted by the presence of high Tumor Mutation Burden (TMB). It is currently believed that other types of tumors may also show the same trend. In lung cancer patients, high nonsynonymous burden (defined as above the median in lung cancer) experienced higher rate of durable clinical benefits, objective response, and progression free survival. High TMB is defined as higher than median or upper third. However, cut-off might vary dependent on the type of tumor. In lung cancer, there is no correlation between PD-L1 expression and TMB. Patients with MSI usually show high TMB. Tumor Mutation Burden (TMB) level (score) may vary dependent on the quality of the DNA and the NGS run. Currently, TMB testing is not standardized. Multiple factors may influence the level of the TMB including percentage of tumor in the analyzed sample, processing of the tissue for paraffin embedding, deamination of the DNA, method of counting mutation rate and quality score of the counted mutations. Clinical and therapy decisions should consider various clinical and laboratory findings. Methodology Nucleic acid is isolated from formalin-fixed paraffin-embedded (FFPE) tissue. Testing is performed using next-generation sequencing (NGS) genes DNA coding regions listed in the Biomarkers Evaluated section of the report. This generally includes sequencing of all the exons. An additional 10 nucleotides at the 5’ and 3’ ends of each coding exon are also sequenced to detect important splicing abnormalities. Sequencing also includes TERT promoter “hotspots” at positions -124 and -146. The NGS method has a typical sensitivity of 3% for detecting common clinically actionable mutations (i.e. “Hotspots”) and 5% for other mutations, however, NRAS p.Q61 has an established limit of detection of 5% mutation allele frequency. For a list of exons with suboptimal coverage in this assay, please contact Client Services (866-776-5907) or refer to our website. Microsatellite instability (MSI) is assessed using 27 markers, which include the five Bethesda consensus microsatellite markers. If a colorectal tumor has less than 30%, but at least 20% tumor, then it is insufficient for MSI by NGS. Therefore, MSI by a PCR-based method will be added. Non-colon tumors will be QNS for MSI (by both NGS and PCR), if they cannot be enriched to at least 30% tumor. If an MSI result is indeterminate by NGS, then reflex to MSI by PCR-based method will be performed, if the minimum requirements for tumor percentage (20% for colorectal and 40% for non-colorectal tumors) and normal tissue control are met. If MSI by PCR is added, then the results will be issued in a separate report. Tumor mutation burden (TMB) is defined as mutations (genomic alterations) per megabase. Genomic alterations include coding single nucleotide variants and InDels that pass a stringent tumor mutation burden protocol. The TMB calculation has been aligned to a reference standard recommended by Friends of Cancer (FOC) Research Tumor Mutation Burden Harmonization Consortium. High TMB is defined as upper quartile of samples tested, while intermediate TMB is aligned to the lowest ranges considered high by other FOC participating laboratories using the FOC reference material. While somatic variant calling is generally concordant with tumor mutation load, there can be variations because the algorithms are different. The TMB method relies on public databases and allele frequency to determine somatic status and must take into account panel bias in order to align to WES results while variant calling relies heavily on manual curation of variantsSAMPLE and more conservatively classifies variants as somatic. Various factors including quantity and quality of nucleic acid, sample preparation, and sample age can affect assay performance. If FISH or IHC testing is ordered, please see individual reports for description of testing.

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Biomarkers Evaluated (by molecular analysis unless otherwise noted) ABL1, ABL2, ACVR1B, AKT1, AKT2, AKT3, ALK, ALK FISH, AMER1 (FAM123B), APC, AR, ARAF, ARFRP1, ARID1A, ARID1B, ARID2, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BCL2, BCL2L1, BCL2L2, BCL6, BCOR, BCORL1, BLM, BRAF, BRAF FISH, BRCA1, BRCA2, BRD4, BRIP1, BTG1, BTK, C11orf30 (EMSY), CARD11, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CD274, CD79A, CD79B, CDC73, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHD2, CHD4, CHEK1, CHEK2, CIC, CREBBP, CRKL, CRLF2, CSF1R, CTCF, CTNNA1, CTNNB1, CUL3, CXCR4, CYLD, DAXX, DDR2, DICER1, DNMT3A, DOT1L, EGFR, EP300, EPCAM, EPHA3, EPHA5, EPHA7, EPHB1, ERBB2, ERBB3, ERBB4, ERG, ERRF11, ESR1, EZH2, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FAS, FAT1, FBXW7, FGF10, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FLT4, FOXL2, FOXP1, FRS2, FUBP1, GABRA6, GATA1, GATA2, GATA3, GATA4, GATA6, GID4 (C17orf39), GLI1, GNA11, GNA13, GNAQ, GNAS, GPR124, GRIN2A, GRM3, GSK3B, H3F3A, HER2 NB FISH, HGF, HIST1H3C, HNF1A, HRAS, HSD3B1, HSP90AA1, IDH1, IDH2, IGF1R, IGF2, IKBKE, IKZF1, IL7R, INHBA, INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KAT6A (MYST3), KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A (MLL), KMT2C (MLL3), KMT2D (MLL2), KRAS, LMO1, LRP1B, LYN, LZTR1, MAGI2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MCL1, MDM2, MDM4, MED12, MEF2B, MEN1, MET, MET FISH, Microsatellite Instability, MITF, MLH1, MPL, MRE11A, MSH2, MSH6, MTOR, MUTYH, MYC, MYC FISH, MYCL (MYCL1), MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKBIA, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NRAS, NSD1, NTRK1, NTRK2, NTRK3, NUP93, PAK3, PALB2, Pan-TRK, PARK2, PAX5, PBRM1, PDCD1LG2, PDGFRA, PDGFRa amp, PDGFRB, PDK1, PD-L1 22C3 FDA (KEYTRUDA®) for Gastric/GEA, PIK3C2B, PIK3CA, PIK3CB, PIK3CG, PIK3R1, PIK3R2, PLCG2, PMS2, POLD1, POLE, PPP2R1A, PRDM1, PREX2, PRKAR1A, PRKCI, PRKDC, PRSS8, PTCH1, PTEN, PTEN FISH, PTPN11, QKI, RAC1, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, RAF1, RANBP2, RARA, RB1, RBM10, RET, RET FISH, RICTOR, RNF43, ROS1, ROS1 FISH, RPTOR, RUNX1, RUNX1T1, SDHA, SDHB, SDHC, SDHD, SETD2, SF3B1, SLIT2, SMAD2, SMAD3, SMAD4, SMARCA4, SMARCB1, SMO, SNCAIP, SOCS1, SOX10, SOX2, SOX9, SPEN, SPOP, SPTA1, SRC, STAG2, STAT3, STAT4, STK11, SUFU, SYK, TAF1, TBX3, TERC, TERT (promoter only), TERT Promoter, TET2, TGFBR2, TNFAIP3, TNFRSF14, TOP1, TOP2A, TP53, TSC1, TSC2, TSHR, Tumor Mutation Burden, U2AF1, VEGFA, VHL, WISP3, WT1, XPO1, ZBTB2, ZNF217, ZNF703

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NCCN Guidelines® are referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Acute Myeloid Leukemia V.1.2016, Breast Cancer V.1.2016, Central Nervous System Cancers V.1.2015, Gastric Cancer V.3.2015, Non-Small Cell Lung Cancer V.4.2016, Colon Cancer V.2.2016, Rectal Cancer V.1.2016, Melanoma V.2.2016, Neuroendocrine Tumors V.1.2015, Ovarian Cancer V.2.2015, Pancreatic Adenocarcinoma V.1.2016, Prostate Cancer V.2.2016, and Uterine Neoplasms V.2.2016. © 2016 National Comprehensive Cancer Network, Inc. All rights reserved. The NCCN Guidelines® and illustrations herein may not be reproduced in any form for any purpose without the express written permission of the NCCN. To view the most recent and complete version of the NCCN Guidelines, go online to NCCN.org

Definitions of Levels of Evidence Tier 1: Variants of Strong Clinical Significance Level A Predictive of response: Therapy is FDA-approved in this disease, based on the presence of this biomarker. Predictive of resistance: Biomarker is included in professional guidelines as providing resistance to therapy. Diagnostic: Biomarker is included in professional guidelines as pathognomonic (required for diagnosis; characteristic of a particular disease). Prognostic: Biomarker is included in professional guidelines for clinical decision-making; specifically, the molecular criteria is included in an accepted, clinically relevant prognostic scoring system.

Level B Predictive of response: Strong evidence (well-powered studies, consensus from experts) that biomarker predicts sensitivity to therapy. Predictive of resistance: Well-powered studies with expert consensus or smaller studies repeatedly confirmed or reproduced by different groups that variant predicts resistance to therapy. Diagnostic: Well-powered studies with expert consensus or repeatedly reported in smaller studies with consistent results or reproduced by different groups indicating diagnostic relevance. These markers may be mentioned in professional guidelines, but are suggestive of, rather than conclusive for, a specific diagnosis. Prognostic: Well-powered studies with expert consensus or smaller studies repeatedly with consistent results or reproduced by different groups indicating prognostic relevance. Level B/C PredictiveSAMPLE of response: Consensus from experts, but lacking well-powered studies that biomarker predicts sensitivity to therapy. Tier 2: Variants of Potential Clinical Significance

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Level C Predictive of response: Therapy is FDA-approved for a different disease, based on the presence of this biomarker; or, criteria for a clinical trial. Predictive of resistance: Preclinical data strongly suggests resistance; reported in clinical cases. Diagnostic: Small studies, diagnostic for a group of related cancers or variants that are supportive of a diagnosis along with other genomic variants. Prognostic: Multiple small studies providing prognostic relevance.

Level C/D Predictive of response: Case reports or small case series including exceptional responders that indicate sensitivity to therapy. Not applicable for drug resistance, prognostic, or diagnostic levels of evidence.

Level D Predictive of response: Plausible sensitivity to therapy based on preclinical studies, which do not need to be disease specific. Predictive of resistance: Limited preclinical data suggesting resistance; no clinical reports. Diagnostic: Small studies or a few case reports support this variant alone or in combination with other biomarkers as assisting diagnosis of this disease. Prognostic: Small studies or a few case reports support this variant alone or in combination with other biomarkers as assisting with prognostic assessment in this disease.

Level E Predictive of response: Poor evidence that biomarker predicts sensitivity to an approved therapy. Not applicable for drug resistance, prognostic, or diagnostic levels of evidence.

Tier 3: Variants of Uncertain Clinical Significance Tier 4: Benign or Likely Benign Variants

Electronic Signature Sample Doctor, M.D. Molecular Pathologist The Technical Component Processing, Analysis and Professional Component of this test was completed at NeoGenomics California, 31 Columbia, Aliso Viejo, CA / 92656 / 866-776-5907 / CLIA #05D1021650 / Medical Director(s): Sally Agersborg, M.D. The performance characteristics of this test have been determined by the performing laboratory. This test has not been approved by the FDA. The FDA has determined such clearance or approval is not necessary. This laboratory is CLIA certified to perform high complexity clinical testing. Images that may be included within this report are representative of the patient but not all testing in its entirety and should not be used to render a result.

SAMPLE

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