Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 1 of 351 PageID #: 7499
`Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 1 of 351 PageID #: 7499
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`EXHIBIT 1
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`EXHIBIT 1
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`Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 2 of 351 PageID #: 7500
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`
`
`IN THE UNITED STATES DISTRICT COURT
`FOR THE DISTRICT OF DELAWARE
`
`
`
`
`
`
`C.A. No. 20-CV-01580-LPS
`
`
`
`) ) ) ) ) ) ) ) )
`
`
`
`GUARDANT HEALTH, INC.,
`
`
`Plaintiff,
`
`
`
`v.
`
`
`FOUNDATION MEDICINE, INC.,
`
`
`Defendant.
`
`
`
`DECLARATION OF JEREMY A. TIGAN IN SUPPORT OF
`FOUNDATION MEDICINE INC.’S BRIEF IN OPPOSITION TO GUARDANT
`HEALTH, INC.’S MOTION FOR A PRELIMINARY INJUNCTION
`
`I, Jeremy A. Tigan, hereby declare:
`
`1.
`
`I am a member of the bar of the State of Delaware and an attorney at the law firm
`
`of Morris, Nichols, Arsht & Tunnell LLP, counsel to Defendant Foundation Medicine, Inc.
`
`(“Foundation Medicine”). I submit this declaration in support of Foundation Medicine’s Brief in
`
`Opposition to Guardant Health, Inc.’s (“Guardant’s”) Motion for a Preliminary Injunction.
`
`2.
`
`To the best of my knowledge and belief, the following appended documents,
`
`Appendix Exhibits 2-147 at A0003-A2704, are true and correct copies of the documents described
`
`in Foundation Medicine’s Appendix in Support of its Brief in Opposition to Guardant’s Motion
`
`for a Preliminary Injunction and the supporting declarations submitted therewith.
`
`3.
`
`I understand Foundation Medicine’s Brief in Opposition to Guardant’s Motion for
`
`a Preliminary Injunction and the supporting declarations submitted therewith refer to pincites by
`
`the last three digits of the applicable production number, where appropriate.
`
`4.
`
`I further understand Appendix Exhibit 75 includes a link in the body of the email.
`
`This link corresponds to Schmitt et al., “Detection of ultra-rare mutations by next-generation
`
`
`
`A0001
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`Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 3 of 351 PageID #: 7501
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`sequencing,” Proc. Natl. Acad. Sci. USA, 2012, 109(36), 14508-14513, Abstract:
`
`https://www.pnas.org/content/early/2012/07/31/1208715109.abstract.
`
`I declare under penalty of perjury that the foregoing is true and correct.
`
`
`
`Dated: February 26, 2021
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`/s/ Jeremy A. Tigan
`Jeremy A. Tigan (#5239)
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`
`
`
`
`2
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`A0002
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`

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`Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 4 of 351 PageID #: 7502
`Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 4 of 351 PageID #: 7502
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`EXHIBIT 2
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`EXHIBIT 2
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`Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 5 of 351 PageID #: 7503
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`Table 1. Companion Diagnostic Indications
`Indication
`Biomarker
`
`Technical Information
` Guardant Health, Inc.
`505 Penobscot Dr.
`Redwood City, CA 94063 USA
`1 Intended Use
` Guardant360® CDx is a qualitative next generation sequencing-based in vitro diagnostic
`device that uses targeted high throughput hybridization-based capture technology for
`detection of single nucleotide variants (SNVs), insertions and deletions (indels) in 55 genes,
`copy number amplifications (CNAs) in two (2) genes, and fusions in four (4) genes.
`Guardant360 CDx utilizes circulating cell-free DNA (cfDNA) from plasma of peripheral
`whole blood collected in Streck Cell-Free DNA Blood Collection Tubes (BCTs). The test is
`intended to be used as a companion diagnostic to identify non-small cell lung cancer
`(NSCLC) patients who may benefit from treatment with the targeted therapy listed in
`Table 1 in accordance with the approved therapeutic product labeling.
`Non-small cell lung
`EGFR exon 19 deletions,
`TAGRISSO® (osimertinib)
`cancer (NSCLC)
`L858R and T790M*
` A negative result from a plasma specimen does not assure that the patient’s tumor is
`negative for genomic findings. NSCLC patients who are negative for the biomarkers listed in
`Table 1 should be reflexed to tissue biopsy testing for Table 1 biomarkers using an FDA-
`approved tumor tissue test, if feasible.
`*The efficacy of TAGRISSO® (osimertinib) has not been established in the EGFR T790M
`plasma-positive, tissue-negative or unknown population and clinical data for T790M
`plasma-positive patients are limited; therefore, testing using plasma specimens is most
`appropriate for consideration in patients from whom a tumor biopsy cannot be obtained.
`Additionally, the test is intended to provide tumor mutation profiling to be used by
`qualified health care professionals in accordance with professional guidelines in oncology
`for cancer patients with any solid malignant neoplasm. The test is for use with patients
`previously diagnosed with cancer and in conjunction with other laboratory and clinical
`findings.
`Genomic findings other than those listed in Table 1 are not prescriptive or conclusive for
`labeled use of any specific therapeutic product.
`Guardant360 CDx is a single-site assay performed at Guardant Health, Inc.
`
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`2 Contraindications
` There are no known contraindications.
` 3 Warnings and Precautions
` Alterations reported may include somatic (not inherited) or germline (inherited)
`alterations. The assay filters germline variants from reporting except for pathogenic
`BRCA1, BRCA2, ATM, and CDK12 alterations. However, if a reported alteration is
`suspected to be germline, confirmatory testing should be considered in the appropriate
`clinical context.
`● The test is not intended to replace germline testing or to provide information about
`cancer predisposition.
`● Somatic alterations in ATM and CDK12 are not reported by the test as they are excluded
`from the test's reportable range.
`● Genomic findings from cfDNA may originate from circulating tumor DNA (ctDNA)
`fragments, germline alterations, or non-tumor somatic alterations, such as clonal
`hematopoiesis of indeterminate potential (CHIP).
`● Allow the tube to fill completely until blood stops flowing into the tube. Underfilling of
`tubes with less than 5 mL of blood (bottom of the label indicates 5 mL fill when tube is
`held vertically) may lead to incorrect analytical results or poor product performance.
`This tube has been designed to fill with 10 mL of blood.
` 4 Limitations
` For in vitro diagnostic use.
`● For prescription use only. This test must be ordered by a qualified medical professional
`in accordance with clinical laboratory regulations.
`● The efficacy of TAGRISSO® (osimertinib) has not been established in the EGFR T790M
`plasma-positive, tissue-negative or unknown population and clinical data for T790M
`plasma-positive patients are limited; therefore, testing using plasma specimens is most
`appropriate for consideration in patients from whom a tumor biopsy cannot be
`obtained.
`● TAGRISSO® efficacy has not been established in patients with EGFR exon 19 deletions <
`0.08% MAF, in patients with EGFR L858R <0.09% MAF, and in patients with EGFR
`T790M < 0.03% MAF.
`● The test is not intended to be used for standalone diagnostic purposes.
`● The test is intended to be performed on specific serial number-controlled instruments
`by Guardant Health, Inc.
`● A negative result for any given variant does not preclude the presence of this variant in
`tumor tissue.
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`● Decisions on patient care and treatment must be based on the independent medical
`judgment of the treating physician, taking into consideration all applicable information
`concerning the patient's condition, such as patient and family history, physical
`examinations, information from other diagnostic tests, and patient preferences, in
`accordance with the standard of care.
`● ctDNA shedding rate may be lower in patients with primary central nervous system
`(CNS) tumors.
` 5 Guardant360 CDx Overview
`
`5.1 Test Summary and Explanation
` Guardant360 CDx is a next generation sequencing-based test for the detection of genetic
`alterations in 55 genes frequently mutated in cancer. It is a companion diagnostic to
`identify non-small cell lung cancer (NSCLC) patients who may benefit from treatment with
`the targeted therapy listed in Table 1 of the Intended Use. Additionally, the test is
`intended to provide tumor mutation profiling to be used by qualified health care
`professionals in accordance with professional guidelines in oncology for cancer patients
`with any solid malignant neoplasm.
`The test report includes variants reported in the following categories (Table 2).
`
`
`Table 2. Category Definitions
`Guardant360 CDx
`Prescriptive use
`for a
`Therapeutic
`Product
`
`Category
`
`Clinical
`Performance
`
`Analytical
`Performance
`
`Category 1:
`Companion
`Diagnostic (CDx)
`Category 2: ctDNA
`Biomarkers with
`Strong Evidence of
`Clinical Significance
`in ctDNA
`08/2020 D-000352 R1 Guardant360 CDx Technical Information
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`Yes
`
`No
`
`Yes
`
`Yes
`
`No
`
`Yes
`
`Comments
`
`ctDNA biomarkers linked to the safe
`and effective use of the corresponding
`therapeutic product, for which
`Guardant360 CDx has demonstrated
`clinical performance shown to
`support therapeutic efficacy and
`strong analytical performance for the
`biomarker.
`ctDNA biomarkers with strong
`evidence of clinical significance
`presented by other FDA-approved
`liquid biopsy companion diagnostics
`for which Guardant360 CDx has
`demonstrated analytical reliability
`but not clinical performance.
`
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`No
`
`No
`
`No
`
`No
`
`ctDNA biomarkers with evidence of
`clinical significance presented by
`Category 3A:
`tissue-based FDA-approved
`Biomarkers with
`companion diagnostics or
`Evidence of Clinical
`professional guidelines for which
`Significance in tissue
`Guardant360 CDx has demonstrated
`supported by: strong
`analytical performance including
`analytical validation
`analytical accuracy, and concordance
`using ctDNA
`of blood-based testing to tissue-based
`testing for the biomarker.
`ctDNA biomarkers with evidence of
`Category 3B:
`clinical significance presented by
`Biomarkers with
`tissue-based FDA-approved
`Evidence of Clinical
`companion diagnostics or
`Significance in tissue
`professional guidelines for which
`supported by:
`Guardant360 CDx has demonstrated
`analytical validation
`minimum analytical performance
`using ctDNA
`including analytical accuracy.
`ctDNA biomarkers with emergent
`evidence based on peer-reviewed
`Category 4: Other
`publications for genes/variants in
`Biomarkers with
`tissue, variant information from well-
`No
`No
`Potential Clinical
`curated public databases, or in-vitro
`Significance
`pre-clinical models, for which
`Guardant360 CDx has demonstrated
`minimum analytical performance.
` 5.2 Sample Collection and Test Ordering
` To order Guardant360 CDx, the Test Requisition Form (TRF) provided with the
`Guardant360 CDx Blood Collection Kit must be fully completed and signed by the ordering
`physician or other authorized medical professional. Refer to the Guardant360 CDx Blood
`Collection Kit Instructions for Use for further details about collecting blood samples and
`shipping samples to the Guardant Health Clinical Laboratory.
`To order the Guardant360 CDx Blood Collection Kit or obtain an electronic version of the
`TRF, contact the Guardant Health Client Services department (Tel: 855.698.8887,
`Fax: 888.974.4258, or Email: clientservices@guardanthealth.com).
` 5.3 Principles of the Procedure
` Guardant360 CDx is performed by a single laboratory, the Guardant Health Clinical
`Laboratory, located in Redwood City, CA, USA. Guardant360 CDx is composed of the
`following major processes:
`● Whole Blood Collection and Shipping
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`Yes
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`● Plasma Isolation and cfDNA Extraction
`● Library Preparation and Enrichment
`● DNA Sequencing
`● Data Analysis and Reporting
` The Guardant360 CDx Blood Collection Kit is used by the ordering laboratories / physicians
`to collect whole blood specimens and ship them to the Guardant Health Clinical Laboratory.
`Whole blood is collected in the provided blood collection tubes, Streck Cell-Free DNA BCTs,
`which stabilize cfDNA and nucleated blood cells for shipping.
`All other reagents, materials and equipment needed to perform the assay are used
`exclusively in the Guardant Health Clinical Laboratory.
`Whole blood specimens are processed in the Guardant Health Clinical Laboratory within 7
`days of blood collection. A minimum of 5 mL whole blood must be received in order to
`achieve optimal performance for the Guardant360 CDx assay. Underfilling of tubes with
`less than 5 mL of blood may lead to incorrect analytical results or poor product
`performance. Plasma is isolated via centrifugation and cfDNA is extracted from plasma.
`cfDNA, 5 to30 ng, is then used to prepare sequencing libraries which are enriched by
`hybridization capture. The enriched libraries are then sequenced using next generation
`sequencing on the Illumina NextSeq 550 platform.
`Sequencing data are then analyzed using a custom-developed bioinformatics pipeline
`designed to detect SNVs, indels, CNAs and fusions from cfDNA. Results (detected or not
`detected) are presented in a results report. A not detected result from a plasma specimen
`for any given variant does not preclude the presence of this variant in tumor tissue.
`The device is designed to detect pre-defined and de novo variants in the genes outlined in
`Table 3. Details on all variants reported can be found in the section 8 Additional
`Guardant360 CDx Variant Details.
`
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`ALK, NTRK1, RET, ROS1
`
`Table 3. Genes Containing Alterations Reported by Guardant360 CDx
`Alteration Type
`Genes
`AKT1, ALK, APC, AR, ARAF, ATM*, BRAF, BRCA1**, BRCA2**, CCND1,
`CDH1, CDK4, CDK6, CDK12*, CDKN2A, CTNNB1, EGFR, ERBB2, ESR1,
`FGFR1, FGFR2, FGFR3, GATA3, GNA11, GNAQ, HRAS, IDH1, IDH2,
`KIT, KRAS, MAP2K1, MAP2K2, MET, MLH1, MTOR, MYC, NF1,
`NFE2L2, NRAS, NTRK1, NTRK3, PDGFRA, PIK3CA, PTEN, RAF1, RET,
`RHEB, ROS1, SMAD4, SMO, STK11, TERT, TSC1, VHL
`AKT1, ALK, APC, ATM*, BRAF, BRCA1**, BRCA2**, CDH1, CDK12*,
`CDKN2A, EGFR, ERBB2, ESR1, FGFR2, GATA3, HNF1A, HRAS, KIT,
`KRAS, MET, MLH1, NF1, PDGFRA, PIK3CA, PTEN, RET, ROS1, STK11,
`TSC1, VHL
`
`Single Nucleotide
`Variants (SNVs)
`Indels
`Copy Number
`Amplifications (CNAs) ERBB2, MET
`Fusions
`*Reporting is enabled for pathogenic germline alterations only. Somatic alterations will not be reported.
`** Reporting is enabled for both germline and somatic alterations.
` 5.4 Reagent, Material, and Equipment Usage
` Reagents, materials, and equipment needed to perform the test are used exclusively in the
`Guardant Health Clinical Laboratory. Guardant360 CDx is intended to be performed with
`the following instruments, as identified by specific serial numbers.
`● Agilent Technologies 4200 TapeStation Instrument
`● Applied Biosystems Veriti 96-Well Thermal Cycler
`● Hamilton Company Microlab STAR
`● Hamilton Company Microlab STARlet
`● Illumina NextSeq 550 Sequencing System
`● Qiagen QIAsymphony SP Instrument
` 6 Summary of Performance Characteristics
`Performance characteristics were established using clinical samples from patients with a
`wide range of cancer types, including those with NSCLC. The clinical samples consisted of
`pools of cfDNA from clinical samples from multiple cancer types, pools of cfDNA from
`clinical samples derived from one cancer type (e.g., samples from patients with NSCLC) or
`un-pooled clinical samples. Studies include CDx variants as well as a broad range of
`representative alteration types (SNVs, indels, CNAs, and fusions) in various genomic
`contexts across a number of genes. Due to limitations in clinical sample availability and
`due to the rarity of the fusions reported by the Guardant360 CDx, contrived samples were
`utilized for some non-clinical studies. A contrived sample functional characterization study
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`a. Concordance - Comparison to NGS Comparator Method
`
`was conducted to demonstrate comparable performance of contrived samples made of cell
`line cfDNA and clinical sample cfDNA so that fusion cell line cfDNA material could be used
`in some non-clinical studies. Fusion positive clinical samples were used to confirm the
`estimated limit of detection, analytical accuracy and precision.
` 6.1 Analytical Accuracy/Concordance
`The detection of alterations by Guardant360 CDx was compared to results of an
`externally validated NGS assay. Samples from 386 donors with different cancer types
`were collected for the study. Sixteen (16) samples failed testing with the comparator
`assay due to instrument failures, while eleven (11) samples failed testing with the
`Guardant360 CDx assay due to an instrument failure due to a power outage. 359
`samples remained comprising three collection sets as follows.
`Collection set one consisted of 100 donor samples selected with the comparator assay
`consecutively without selection for any specific variants. Since the first sample
`collection was expected to lack many rare variants, in the second collection set, a set of
`100 positive samples were selected with the comparator assay. Collection set three
`consisted of 159 samples selected from the Guardant Health biobank based on
`Guardant360 LDT results to include additional rare variants including gene fusions
`which were not available from collection sets 1 and 2.
`Of 359 patients, no samples failed QC on Guardant360 CDx, and three samples failed
`with the comparator NGS assay. In total, 356 donor samples across 18 cancer types,
`which all passed every QC metric were used for the concordance analysis. The cancer
`types represented in this study included lung (178), gastrointestinal (82), colon (25),
`breast (17), head and neck (13), prostate (12), genitourinary (7), bladder (3), stomach
`(3), pancreas (3), endocrine (2), liver (2), ovarian (2), kidney (2), gynecologic (1),
`esophagus (1), skin (1), and other (5). A summary of Positive Percent Agreement (PPA)
`and Negative Percent Agreement (NPA) with 95% confidence intervals (CI) is provided
`in Table 4 for CDx alterations in samples from the intended use population, i.e., 176
`patients with NSCLC. Agreement rates for each of the CDx variants ranged from 95% to
`100% for PPA, and from 98.1% to 99.9% for NPA. The reported PPA and NPA were not
`adjusted for the distribution of samples from collection set 3 selected using Guardant
`LDT results. A summary of PPA and NPA for other clinically significant variant
`categories and for panel wide for SNVs and indels over all sample collections is
`provided in Table 4.
`Positive agreement rates were evaluable for nine (9) patients with clinical Category 2
`variants, which consisted of clinically relevant PIK3CA mutations in breast cancer
`patients that included E545A, E542K, E545K, H1047R, and H1047L variants.
`Concordance analysis resulted in 100% PPA and 100% NPA for the Category 2 variants.
`Positive agreement rates for clinical Categories 3 and 4 variants resulted in 93.5% PPA
`and 86.1% PPA, respectively. Variants in clinical category 3 and 4 showed 99.8% and
`100.0% NPA.
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`MET amplifications had a PPA of 56%, which is attributed to differences in reporting of
`copy number alterations by the Guardant360 CDx and the comparator assay. The
`Guardant360 CDx reports on only focal amplifications and not chromosome-arm
`amplifications, while the NGS comparator assay reports all amplifications.
`The study demonstrated a PPA of 82.5% for indels, 91.4% for SNVs and >99% NPA for
`the entire reportable range, i.e., panel-wide, demonstrating the analytical accuracy of
`the device.
`
`
`Table 4. Summary of Concordance Between Guardant360 CDx and NGS
`Comparator Method
`
`Comparator (+)
`
`Guardant360
`
`CDx(-),
`
`Comparator (-)
`
`Guardant360
`
`CDx(+),
`
`Comparator (+)
`
`Guardant360
`
`CDx(+),
`
`Alteration Type
`
`1
`153
`1
`3
`19
`EGFR T790M
`1
`157
`0
`1
`18
`EGFR L858R
`EGFR exon 19
`6
`1024
`1
`1
`30
`deletions
`Category 2
`5
`76
`0
`0
`9
`Variants
`Category 3
`50
`6191
`8
`11
`115
`Variants
`Category 4
`388
`137582
`68
`58
`420
`Variants
`1
`330
`10
`3
`13
`MET CNAs
`1
`339
`2
`0
`15
`ERBB2 CNAs
`1
`351
`0
`0
`5
`NTRK1 Fusions
`1
`342
`1
`2
`11
`RET Fusions
`ALK Fusions
`10
`2
`0
`344
`1
`ROS1 Fusions
`11
`0
`0
`345
`1
`08/2020 D-000352 R1 Guardant360 CDx Technical Information
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`PPA
` (95% CI)
`
`95.0%
`(75.1%, 99.9%)
`100.0%
`(81.5%, 100.0%)
`96.8%
`(83.3%, 99.9%)
`100.0%
`(66.4%,100.0%)
`93.5%
`(87.6%, 97.2%)
`86.1%
`(82.7%, 89.0%)
`56.5%
`(34.5%, 76.8%)
`88.2%
`(63.6%, 98.5%)
`100.0%
`(47.8%, 100.0%)
`91.7%
`(61.5%, 99.8%)
`100.0%
`(69.2%, 100.0%)
`100.0%
`
`NPA
`(95% CI)
`
`98.1%
` (94.5%, 99.6%)
`99.4%
`(96.5%, 100.0%)
`99.9%
`(99.5%, 99.9%)
`100.0%
`(95.3%, 100.0%)
`99.8%
` (99.7%, 99.9%)
`100.0%
`(99.9%, 100.0%)
`99.1%
` (97.4%, 99.8%)
`100.0%
`(98.9%, 100.0%)
`100.0%
`(98.9%, 100.0%)
`99.4%
`(97.9%, 99.9%)
`99.4%
` (97.9%, 99.9%)
`100.0%
`
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`Patients (n)
`
`176
`176
`176
`17
`N/A*
`356
`356
`356
`356
`356
`356
`356
`
`Variants (n)
`
`Possible
`
`Comparator (-)
`
`Guardant360
`
`CDx(-),
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`A0010
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`(98.9%, 100.0%)
` (71.5%,100.0%)
`99.9%
`91.5%
`13726844
`356
`38560
`40
`48
`428
`Panel-Wide SNVs
`(99.9%, 99.9%)
`(88.5%, 93.8%)
`99.9%
`82.5%
`15717238
`356
`44150
`25
`19
`118
`Panel-Wide Indels
`(99.9%, 99.9%)
`(75.3%, 88.4%)
` * For Category 3, no number is given. This is because Category 3 is a merge of many different variants, each
`with a specific set of cancer types that qualify the variant to belong in Category 3. This means that a different
`number of patients was associated with each variant within Category 3. For this level, the concordantly
`negative population was computed as the sum of the concordantly negative populations if each variant in this
`category was treated independently.
` 6.2 Contrived Sample Functional Characterization (CSFC) Study
`A CSFC study was performed to demonstrate comparable performance between
`contrived samples that consisted of fusion cell line cfDNA material and fusion positive
`clinical sample cfDNA material. The CSFC study was performed using 5 ng DNA input
`(the lowest cfDNA input for the assay) to compare the performance of the Guardant360
`CDx with cfDNA derived from cell lines and cfDNA derived from multiple clinical
`samples from multiple cancer types with ALK, NTRK1, RET, and ROS1 fusions. The cell
`line and clinical cfDNA sample pools contained known fusion events that were diluted
`with pools of wild-type (WT) cfDNA from multiple clinical specimens from multiple
`cancer types to pre-determined MAF levels (targeted levels were above and below LoD;
`see Table 5). Cell line cfDNA sample pools were tested across 13-20 replicates, 13
`replicates for level 6, 14 replicates for level 2, and 20 replicates for the other levels at 5
`ng cfDNA input. Clinical cfDNA sample pools from multiple cancer types were tested
`with 14 replicates at 5 ng cfDNA input. Both cell line and clinical cfDNA sample pools
`were tested with an orthogonal method to confirm MAF level. Detection rates of the 4
`fusions, for each titration level, and for each of the two types of pools, are presented in
`Table 5.
`Based on these analyses, the results demonstrate that the performance of the
`Guardant360 CDx is similar for both fusion positive contrived cfDNA samples and for
`fusion positive clinical cfDNA samples.
`
`Detection Rate (95% confidence interval)
`Fusion
`Type
`0.07%
`0.175%
`0.35%
`EML4-ALK
`Cell line
`5.0%
`28.6%
`50.0%
`(0.1%,
`(8.4%,
`(27.2%,
`24.9%)
`58.1%)
`72.8%)
`08/2020 D-000352 R1 Guardant360 CDx Technical Information
`
`1.8%
`100.0%
`(75.3%,
`100%)
`
`
`9 of 63
`
`Level 6
`Target
`MAF
`
`Level 4
`Target
`MAF
`
`0.7%
`90.0%
`(68.3%,
`98.8%)
`
`Level 5
`Target
`MAF
`
`1.4%
`100.0%
`(83.2%,
`100.0%)
`
`
`Table 5. Fusion Detection Rate in the CSFC study
`Sample
`
`Level 1
`Target
`MAF
`
`Level 2
`Target
`MAF
`
`Level 3
`Target
`MAF
`
`A0011
`
`

`

`Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 14 of 351 PageID #: 7512
`
`ROS1-
`
`PLEKHA6-
`
`a. Limit of Blank (LoB)
`
`7.1%
`28.6%
`50.0%
`85.7%
`Clinical
`EML4-ALK
`(8.4%,
`(23.0%,
`(57.2%,
`(0.2%,
`33.9%)
`58.1%)
`77.0%)
`98.2%)
`15.0%
`35.7%
`80.0%
`95.0%
`Cell line
`CCDC6- RET
`(3.2%,
`(12.8%,
`(56.3%,
`(75.1%,
`37.9%)
`64.9%)
`94.3%)
`99.9%)
`7.1%
`14.3%
`64.3%
`85.7%
`TRIM33- RET Clinical
`(0.2%,
`(1.8%,
`(35.1%,
`(57.2%,
`33.9%)
`42.8%)
`87.2%)
`98.2%)
`0.0%
`21.4%
`50.0%
`75.0%
`Cell line
`(0.0%,
`(4.7%,
`(27.2%,
`(50.9%,
`SLC34A2
`16.8%)
`50.8%)
`72.8%)
`91.3%)
`7.1%
`42.9%
`85.7%
`100.0%
`ROS1- CD74
`Clinical
`(0.2%,
`(17.7%,
`(57.2%,
`(76.8%,
`33.9%)
`71.1%)
`98.2%)
`100.0%)
`15.0%
`50.0%
`40.0%
`90.0%
`TPM3-NTRK1 Cell line
`(3.2%,
`(23.0%,
`(19.1%,
`(68.3%,
`77.0%)
`63.9%)
`98.8%)
`37.9%)
`21.4%
`35.7%
`85.7%
`100.0%
`Clinical
`(4.7%,
`(12.8%,
`(57.2%,
`(76.8%,
`NTRK1
`50.8%)
`64.9%)
`98.2%)
`100.0%)
`ND: Not determined
` 6.3 Analytical Sensitivity
`The LoB was established by evaluating whole blood samples from healthy age-matched
`donor samples. sixty-two (62) donor samples confirmed to be mutation negative based
`on sequencing with an externally validated orthogonal method were processed using
`30 ng of cfDNA input with the Guardant360 CDx (highest DNA input for the assay)
`across three lots of reagents, operator groups, and instruments. Of the 62 donor
`samples, 58 donor samples were tested with 4 replicates, while 4 donors were tested
`with 2 replicates for a total of 240 replicates analyzed to assess the false positive rate of
`Guardant360 CDx. This study demonstrated a near zero false positive rate across the
`entire reportable range, as shown in Table 6. The false positive rate was zero for
`Category 1 (CDx) and Category 2 variants.
`Category
`Positive Rate
`Positive Rate
`Category 1: EGFR L858R
`0%
`0 (0/240)
`Category 1: EGFR T790M
`0%
`0 (0/240)
`08/2020 D-000352 R1 Guardant360 CDx Technical Information
`
`
`Table 6. LoB Study Summary Results
`Per Position False
`
`100.0%
`(76.8%,
`100.0%)
`100.0%
`(83.2%,
`100.0%)
`100.0%
`(76.8%,
`100.0%)
`100%
`(83.2%,
`100.0%)
`100.0%
`(83.9%,
`100.0%)
`100.0%
`(83.2%,
`100.0%)
`ND
`
`100.0%
`(76.8%,
`100.0%)
`100.0%
`(75.3%,
`100.0%)
`100.0%
`(76.8%,
`100.0%)
`100.0%
`(75.3%,
`100%)
`ND
`100.0%
`(75.3%,
`100.0%)
`100.0%
`(76.8%,
`100.0%)
`
`Per Sample False
`
`
`
`
`
`10 of 63
`
`A0012
`
`

`

`Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 15 of 351 PageID #: 7513
`
`
`b. Limit of Detection (LoD)
`
`0 (0/240)
`0%
`Category 1: EGFR Exon 19 deletions
`0 (0/240)
`0%
`Category 2
`<0.00005%
`1.67% (4/240)
`Panel-wide SNVs (38,560 bp)
`(4/(38,560*240))
`<0.00002%
`0.83% (2/240)
`Panel-wide Indels (44,150 bp)
`(2/(44,150*240))
`0.42% (1/240)
`0.2% (1/(2*240))
`Panel-wide CNAs (2 genes)
`0 (0/240)
`0%
`Panel-wide Fusions (4 genes)
`The LoD for the Guardant360 CDx variants with CDx claims, representative SNVs and
`indels, and all reportable CNAs and fusions was established at the lowest and highest
`claimed cfDNA input amounts (5 and 30ng). LoD established for fusions using cfDNA
`derived from cell lines was confirmed at 5ng cfDNA input using cfDNA derived from
`clinical patient samples. LoDs were further confirmed in the clinical pools of relevant
`cancer types for CDx variants and additional representative variants, including long
`indels and homopolymers in a combined LoD confirmation and precision study.
`For SNVs, indels, including CDx variants and for CNAs, the Guardant360 CDx LoD was
`established by combining cfDNA from clinical plasma samples from multiple cancers to
`create pools of material comprising multiple known alterations. The LoD was
`established with these clinical cfDNA sample pools at 5ng and 30ng input, using a
`combination of probit and empirical approaches. Samples were titrated at 5 different
`MAF values that included levels above and below the LoD for SNVs, and indels or copy
`numbers values for CNAs and tested across 20 replicates for 5 ng input and 14
`replicates for 30 ng input across two reagent lots.
`The LoDs of three (3) CDx alterations representing EGFR T790M, EGFR L858R, and
`EGFR exon 19 deletions established using pools of cfDNA from clinical plasma samples
`from multiple cancer types are summarized in Table 7. The LoD was confirmed for CDx
`variants using cfDNA sample pools from patients with NSCLC only; refer to Table 9
`below.
`
`
`Table 7. Summary of Established LoD for Alterations Associated with CDx Claims
`using Pools of cfDNA from Clinical Plasma Samples from Multiple Cancer Types
`Alteration Type
`Alteration
`LoD (5ng input)
`LoD (30 ng input)
`
`0.2% MAF
`0.2% MAF
`0.2% MAF
`
`
`
`11 of 63
`
`SNV
`EGFR T790M
`SNV
`EGFR L858R
`EGFR exon 19 deletion
`Indel (15 bp)
`
`08/2020 D-000352 R1 Guardant360 CDx Technical Information
`
`1.1% MAF
`1.0% MAF
`1.5% MAF
`
`A0013
`
`

`

`Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 16 of 351 PageID #: 7514
`
`Alteration
`
`LoD, 30 ng
`
`Table 8. LoD Establishment Study Summary Results for Representative Variants
`using Pools of cfDNA Clinical Plasma Samples from Multiple Cancer Types
`LoD, 5 ng
`
`The LoD estimates for SNV, indels, and CNA alterations established using pools of cfDNA
`from clinical plasma samples from multiple cancer types are summarized in Table 8.
`For fusions, the Guardant360 CDx LoD was established using cfDNA from cell lines with
`known fusions titrated into wild-type (WT) cfDNA from clinical plasma samples.
`Samples were titrated at 5 different MAF values for fusions across 20 replicates for 5 ng
`cfDNA input and 14 replicates for 30 ng cfDNA input across two reagent lots. The
`established LoD was then confirmed using fusion positive cfDNA from clinical plasma
`samples at 5 ng cfDNA input only. Fusion positive cfDNA from clinical samples were
`titrated across 5 concentrations with 14 replicates across 2 reagent lots.
`The higher of the LoD values established using cell lines and confirmed using clinical
`samples were used to claim the LoD performance levels of the test for fusions at 5 ng
`(Table 8).
`
`Alteration Type
`(MAF/CN)
`(MAF/CN)
`BRAF V600E
`0.2%
`1.8%
`SNV
`KRAS G12V
`0.5%
`1.5%
`SNV
`NRAS Q61R
`0.8%
`3.0%
`SNV
`BRCA1 p.E23fs
`0.8%
`2.6%
`Indel (2 bp)
`BRCA2 p.S1982fs
`0.4%
`1.3%
`Indel (1 bp)
`EGFR exon 20 insertion,
`0.2%
`0.8%
`Indel (9 bp)
`p.Ala767_Val769dup
`ERBB2 exon 20 insertion,
`0.2%
`1.1%
`Indel (12 bp)
`p.A775_G776insYVMA
`MET
`2.4
`2.4
`CNA
`ERBB2
`2.3
`2.3
`CNA
`(0.2%)
`0.9% (0.9%)
`Fusion
`(0.1%)
`1.1% (0.7%)
`Fusion
`(0.2%)
`1.9% (1.2%)
`Fusion
`(0.2%)
`1.4% (1.5%)
`Fusion
`Note: Numbers in parentheses represent LoD established using cell line derived cfDNA.
`MAF: Mutant Allele Fraction, CN: copy number
`The established LoD was confirmed for CDx variants by testing clinical patient pools
`exclusively from NSCLC patients targeting 1-1.5x LoD of the established LoD (refer to
`Table 9) across at least 20 replicates at 5 ng input using a combined LoD Confirmation
`and Precision Study. Similarly, the established LoD was confirmed for SNVs and indels
`
`08/2020 D-000352 R1 Guardant360 CDx Technical Information
`
`
`12 of 63
`
`NTRK1
`RET
`ROS1
`ALK
`
`
`
`A0014
`
`

`

`Case 1:20-cv-01580-LPS Document 39-1 Filed 03/05/21 Page 17 of 351 PageID #: 7515
`
`Number
`Positive /
`Number
`Expected
`
`20/20
`19/20
`20/20
`20/20
`20/20
`19/20
`21/21
`21/21
`22/22
`20/20
`21/21
`20/20
`20/20
`
`Table 9. Combined LoD Confirmation and Precision Study Summary Results for
`CDx Variants and Representative Variants
`
`in clinical pools made exclusively from the relevant cancer type source material
`prepared with 5 ng cfDNA input targeting 1-1.5x LoD and run in