Oncomine Dx Express Test

The Oncomine Dx Express Test reports the following genes by alteration type from DNA and RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples: 

Table 1: Oncomine Dx Express Test FFPE gene list

The Oncomine Dx Express Test reports the following genes by alteration type from cell free total nucleic acid (cfTNA) extracted from plasma samples:

Table 2: Oncomine Dx Express Test plasma gene list

Analytical accuracy study

The analytical accuracy was evaluated with 151 clinical FFPE samples from six cancer types (breast, colorectal, glioma, melanoma, non-small cell lung, and thyroid cancer) using the Oncomine Dx Express Test and two NGS-based orthogonal reference assays. The concordance evaluation study was performed at two sites.

The positive percent agreement (PPA) and negative percent agreement (NPA) were defined as the proportion of variant-positive and variant-negative specimens, respectively, as determined by the reference methods that were also determined by the Oncomine Dx Express Test.

Analytical accuracy results are summarized in the following table:

Table 3. Oncomine Dx Express Test FFPE concordance

Limit of Blank study

The limit of blank was established by profiling 30 clinical FFPE samples confirmed to be variant-negative by a reference method. The study included: 2 replicates per sample, 2 reagent lots, and 11 tissue types (bladder, brain, breast, bile duct, colon, endometrium, lung, pancreas, prostate, skin, and thyroid).

For all 30 samples, the false-positive rate of the test was determined to be 0.75% for SNVs, 0% for indels, 0% for CNVs, and 0% for fusions. By definition of the Clinical and Laboratory Standards Institute (CLSI) EP17-A2, the limit of blank is zero.

Limit of Detection (LoD) study

The LoD was evaluated with 20 representative SNVs, indels, CNVs, and RNA fusions detected by the Oncomine Dx Express Test in clinical FFPE samples. The LoD is defined as the lowest variant level that can be detected at least 95% of the time.

Clinical specimens representing six cancer types (breast cancer, colorectal cancer, glioma, NSCLC, melanoma, and thyroid cancer) were used as the source of DNA and RNA. Variant-containing specimens were blended with wild-type samples, and the study included: 6 titration levels, 2 reagent lots, and 10 replicates per sample blend.

Based on a representative approach, the LoDs ranged from:

• 3.07% to 6.48% allelic frequencies for SNVs and indels (mean = 4.29% allelic frequency)
• 4.91 to 5.32 copies for CNVs
• 5.27 to 12.35 molecular counts (median = 8.85 molecular counts) and 7.87 to 207.5 reads for fusions

Precision study

The repeatability and reproducibility were evaluated using 20 representative DNA variants and RNA fusions in FFPE samples from six cancer types: breast cancer, colorectal cancer, glioma, melanoma, NSCLC, and thyroid cancer.

Three sites, with two operators and instruments per site, were used for the study. DNA and RNA was extracted from clinical FFPE samples, then blended with wild-type DNA or RNA into seven DNA blends and seven RNA blends. Two levels per blend were generated and distributed to sites and operators for testing.

The mean call rates excluding no-calls were 99.23%, 100%, and 99.69% for variant-positive SNVs/indels, CNVs, and fusions, respectively. The mean call rates excluding no-calls was 100% for wild-type DNA (negative-calls) and wild-type RNA.

Analytical accuracy study

The analytical accuracy of the Oncomine Dx Express Test for plasma was evaluated with 80 plasma samples from NSCLC comprising 40 variant-positive and 40 variant-negative samples (Table 4). The concordance study was performed at two sites that received an identical set of samples. One site used the Oncomine Dx Express Test, and the second site used an NGS-based reference assay.

The PPA and NPA were defined as the proportion of variant-positive and variant-negative specimens, respectively, as determined by the reference method and the Oncomine Dx Express Test analytical accuracy results are summarized in the following table:

Table 4. Oncomine Dx Express Test plasma concordance

Limit of Blank study

The limit of blank was established by profiling cfTNA extracted from 30 blood plasma samples from healthy donors confirmed to be variant-negative by a reference method. The study included two replicates per sample and two reagent lots.

For all 30 samples, the false positive rate was determined to be 0.20% for SNVs, 0% for indels, and 0% for fusions. By definition of CLSI EP17-A2, the limit of blank is zero.

Limit of Detection (LoD) study

The LoD was evaluated with 11 representative SNVs, indels, CNVs, and RNA fusions detected by the Oncomine Dx Express Test in clinical plasma samples. The LoD is defined as the lowest variant level that can be detected at least 95% of the time. The study included 6 titration levels, 2 reagent lots, 10 replicates per sample blend, and 2 cfTNA input levels (5 ng and 30 ng).

Based on a representative variant approach, the LoDs for SNVs and indels range from 0.65% to 1.82% allelic frequency (mean = 1.9% allelic frequency) for the 5 ng input level. The LoDs for SNVs and indels at the 30 ng input level ranged from 0.31% to 0.42% allelic frequency (mean = 0.36% allelic frequency).

The LoDs for RNA fusions at the 5 ng input level ranged from 9.9 to 19.6 molecular counts (median = 14.3 molecular counts). The LoDs for RNA fusions at the 30 ng input level ranged from 6.4 to 8.0 molecular counts (median = 7.5 molecular counts).

Precision study

The repeatability and reproducibility were evaluated using contrived cfTNA plasma samples prepared by blending cfTNA extracted from variant-positive cell lines and cfTNA from healthy donor plasma.

Three sites, with two operators and instruments per site, were used for the study. Site 1 had four instruments, and sites 2 and 3 had two instruments each. The mean call rates excluding no-calls were 99.86%, and 99.25% for variant-positive SNVs/indels and fusions, respectively.

The mean call rates excluding no-calls was 100% for wild-type DNA (negative-calls) and wild-type RNA.