Search Results

The Tempus xR IVD assay is a qualitative next generation sequencing-based in vitro diagnostic device that uses targeted high throughput hybridization-based capture technology for detection of rearrangements in two genes, using RNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens from patients with solid malignant neoplasms.

Information provided by xR IVD is intended to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with previously diagnosed solid malignant neoplasms. Results from xR IVD are not intended to be prescriptive or conclusive for labeled use of any specific therapeutic product.

xR IVD is a next generation sequencing (NGS)-based assay for the detection of alterations from RNA that has been extracted from routinely obtained FFPE tumor samples. Extracted RNA undergoes conversion to double stranded cDNA and library construction, followed by hybridization-based capture using a whole-exome targeting probe set with supplemental custom Tempus-designed probes. Using the Illumina® NovaSeq 6000 platform qualified by Tempus, hybrid-capture–selected libraries are sequenced, targeting > 6 million unique deduplicated reads. Sequencing data is processed and analyzed by a bioinformatics pipeline to detect gene rearrangements, including rearrangements in BRAF and RET.

Alterations are classified for purposes of reporting on the clinical report as Level 2 or Level 3 alterations in accordance with the FDA Fact Sheet describing the CDRH's Approach to Tumor Profiling for Next Generation Sequencing Tests and as follows:

• Level 2: Genomic Findings with Evidence of Clinical Significance
• Level 3: Genomic Findings with Potential Clinical Significance

xR IVD is intended to be performed with the following key components, each qualified and controlled by Tempus under its Quality Management System (QMS):

• Reagents
• Specimen Collection Box
• Software
• Sequencing Instrumentation

1. Reagents

All reagents used with respect to the operation of xR IVD are qualified by Tempus.

2. Test Kit Contents

xR IVD includes a specimen collection and shipping box (the Specimen Box). The Specimen Box contains the following components:

• Informational Brochure with Specimen Requirements
• Collection Box Sleeve
• Collection Box Tray
• Seal Sticker
• ISO Label

3. Software

The proprietary xR IVD bioinformatics pipeline comprises data analysis software necessary for the xR IVD assay (software version is displayed on the xR IVD clinical report). The software is used with sequence data generated from NovaSeq 6000 instruments qualified by Tempus. Data generated from the pipeline is saved to a cloud infrastructure.

4. Instrument

xR IVD uses the Illumina NovaSeq 6000 Sequencer, a high throughput sequencing system employing sequencing-by-synthesis chemistry. The xR IVD device is intended to be performed with serial number-controlled instruments. All instruments are qualified by Tempus utilizing the Tempus Quality Management System (QMS).

5. Sample preparation

FFPE (Formalin Fixed Paraffin Embedded) tumor specimens are received either as unstained tissue sections on slides or as an FFPE block using materials supplied in the Specimen Box and prepared following standard pathology practices. Preparation and review of a Hematoxylin and Eosin (H&E) slide is performed prior to initiation of the xR IVD assay. H&E stained slides are reviewed by a board-certified pathologist to ensure that adequate tissue, tumor content and sufficient nucleated cells are present to satisfy minimum tumor content (tumor purity).

Specifically, the minimum recommended tumor purity for detection of alterations by xR IVD is 20%, with macrodissection required for specimens with tumor purity lower than 20%. The recommended tumor size and minimum tumor content needed for testing are shown in Table 1, below.

Table 1: Tumor Volume and Minimum Tumor Content

*These requirements are based on the specimen's tumor content

6. RNA extraction

Nucleic acids are extracted from tissue specimens using a magnetic bead-based automated methodology followed by DNAse treatment. The remaining RNA is assessed for quantity and quality (sizing) at RNA QC1, which is a quality check (QC) to ensure adequate RNA extraction. The minimum amount of RNA required to perform the test is 50 ng. RNA is fragmented using heat and magnesium, with variable parameters, to yield similar sized fragments from RNA inputs with different starting size distributions.

7. Library preparation

Strand-specific RNA library preparation is performed by synthesizing the first-strand cDNA using a reverse transcriptase (RT) enzyme followed by second-strand synthesis using a DNA polymerase to create double stranded cDNA. Adapters are ligated to the cDNA and the adapter-ligated libraries are cleaned using a magnetic bead-based method. The libraries are amplified with high fidelity, low-bias PCR using primers complementary to adapter sequences. Amplified libraries are subjected to a 1X magnetic bead based clean-up to eliminate unused primers, and quantity is assessed (QC2) to ensure that pre-captured libraries were successfully prepared. Each amplified sample library contains a minimum of 150 ng of cDNA to proceed to hybridization.

8. Hybrid capture

After library preparation and amplification, the adapter-ligated library targets are captured by hybridization, clean-up of hybridized targets is performed, and unbound fragments are washed away. The captured targets are enriched by PCR amplification followed by a magnetic bead-based clean-up to remove primer dimers and residual reagents. To reduce non-specific binding of untargeted regions, human COT DNA and blockers are included in the hybridization step. Each post-capture library pool must satisfy a minimum calculated molarity (≥2.7 nM) to proceed to sequencing (QC3). The molarity is used to load the appropriate concentration of library pools onto sequencing flow cells.

9. Sequencing

The amplified target-captured libraries are sequenced with a 2x76 read length to an average of 50 million total reads on an Illumina NovaSeq 6000 System using patterned flowcells (SP/S1, S2, or S4). Pooled sample libraries are fluorometrically quantified and normalized into a sequencing pool of up to 28 samples (SP flowcell), 56 samples (S1 flowcell), 140 samples (S2 flowcell), 336 samples (S4 flowcell) with each flowcell including 2 external controls. Partial batches are supported using a set threshold of loading capacity down to a defined percentage. Pooled sample libraries are loaded on a sequencing flow cell and sequenced.

10. Data Analysis

a. Data Management System (DMS): Sequence data is automatically processed using software that tracks sample names, sample metadata processing status from sequencing through to analysis and reporting. Reports of identified alterations are available in a web-based user interface for download. Sequencing and sample metrics are available in run and case reports, including sample and sequencing quality.

b. Demultiplexing and FASTQ Generation: Demultiplexing software generates FASTQ files containing sequence reads and quality scores for each of the samples on a sequencing run. The FASTQ formatted data files are used for subsequent processing of samples.

c. Indexing QC Check: Samples are checked for an expected yield of sequence reads identified to detect mistakes in pooling samples. Samples outside the expected range are marked as failed.

d. Read Alignment and BAM Generation: Genome alignment is performed to map sequence reads for each sample to the human reference genome (hg19). Alignments are saved as Binary Alignment Map (BAM) formatted files, which contain read placement information relative to the reference genome with quality scores. Aligned BAM files are further processed in a pipeline to identify genomic alterations.

e. Sample QC check: A sample QC check (QC4) evaluates the quality of the samples processed through the bioinformatics pipeline (sample level metrics in Table 2). Samples are evaluated for contamination by evaluating the percent of a tumor sample contaminated with foreign nucleic acid with a threshold below 5%. Sample sequencing coverage is assessed through RNA gene-ids expressed which counts all genes raw expression abundance (>12,000) and RNA GC-distribution (45-59%). The sample mapping rate (>80%), RNA strand % sense (>88%) and RNA strand % failed (≤ 10%) metrics provide confidence in the sample quality.

f. Alteration calling: A fully automated pipeline for bioinformatic analysis is used to identify gene rearrangements. The assay is validated to report specific gene rearrangements. Gene rearrangements are identified based on observations of reads supporting gene rearrangements in genomic alignments of discordantly mapped or split read pairs.

11. Controls

a. Negative control: A no template control (NTC) is processed to serve as a negative control to validate the acceptability of all the test samples processed through extraction, library preparation and hybridization and capture steps by testing for sample or reagent contamination. The NTC is not included on the sequencing run.

b. Positive control: xR IVD uses multiple external controls consisting of contrived material with synthetically derived alterations or a pool of multiple cell lines. A positive control sample containing known gene rearrangements will be included with each sequencing run. The external controls are processed from library preparation through sequencing to serve as an end to end control to demonstrate assay performance. The external controls are checked during library preparation and after sequencing. Failure of the external control to meet the pre-defined quality metrics will result in all test samples on the run being reported as Quality Control (QC) failure.

12. Result reporting

xR IVD reports oncologically relevant gene rearrangements as genomic findings with evidence of clinical significance or with potential clinical significance. Gene rearrangements are assessed as oncogenic based on required genomic regions specified in a Tempus-developed curated database. Gene rearrangements that retain the genomic region(s) required for oncogenicity are assigned a level of clinical significance consistent with FDA's Fact Sheet and reported. Gene rearrangements that do not retain the region(s) required for oncogenicity are not reported.

13. Quality metrics

Reporting takes into account the quality metrics outlined in Table 2. Quality metrics are assessed across the following categories:

• Batch-level: Metrics that are quantified per sequencing run; if the positive control fails these criteria, no results are reported for the entire batch of samples.
• Sample-level: Metrics that are quantified per sample; no device results are generated for samples failing these metrics. These metrics are also referred to as sequencing quality control (QC4).
• Analyte-level: Metrics that are quantified for individual alteration types. Alterations passing analyte-level metrics (threshold) are reported.

Table 2: Summary of xR IVD Post-Sequencing Key Quality Metrics at Batch, Sample (QC4), and Analyte Levels

N/A

Ask a specific question about this device