For Research Use Only. Not for use in diagnostic procedures.

Performance of the AVENIO Tumor Tissue Analysis Kits with Core Needle Biopsy (CNB) and Fine Needle Aspiration (FNA) Samples

Introduction

Materials and Methods

The AVENIO Tumor Tissue Analysis Kits (Research Use Only, Not for use in diagnostic procedures) have been validated using surgical formalin-fixed, paraffin-embedded (FFPE) samples, but many archival FFPE specimens have limited tissue material available when they are derived from biopsies such as core needle biopsy (CNB) and fine needle aspiration (FNA). In this study, we demonstrated the feasibility of utilizing CNB and FNA FFPE samples with the AVENIO Tumor Tissue Analysis Kits.

Samples84 tissue samples obtained using commonly employed minimally invasive CNB and FNA procedures were analyzed. Of the total 84 samples, 59 samples (70%) were CNBs and 25 samples (30%) were FNAs. Sample tumor content in CNB and FNA samples ranged between 2% - 80% and 8% - 70%, respectively. Sample size ranged between 1-72 mm² and 36-060 mm², for CNB and FNA samples respectively. Out of 84 specimens, 78 tissue samples (93%) were obtained from lung lesions, including non–small cell lung cancer (NSCLC), squamous cell carcinoma, large cell carcinoma, and small cell lung cancer (SCLC), while 6 tissue samples (7%) were obtained from colorectal cancer (CRC).

Sample Collection ProcedureCore needle biopsy was performed with a needle biopsy apparatus of varying sizes between 14 and 20 gauge from target lesions. The CNB samples were processed through 10% neutral buffered formalin as routine clinical specimens. About 2-5 cores or multiple, uncountable clustered cores were embedded in a single paraffin block. The paraffin-embedded block was sectioned and stained with routine hematoxylin-eosin (H&E) stains.

Fine needle aspiration specimens were collected from target lesions using a needle biopsy apparatus with varied sizes between 19 and 25 gauge and transferred to a container with 10% neutral buffered formalin. The cells were dispersed in the fluid. An aliquot of the suspension was selected for further processing. The cells were spread on one or multiple slides as a thin layer by sedimentation.

AVENIO Sample Prep and Workflow Modifications CNB and FNA samples were used as sample input into the AVENIO Tumor Tissue Analysis workflow using the Expanded Kit according to the user manual with the following modifications. For CNB samples, ten to fifteen 20 µm curls were used according to pathologist’s recommendation based on tissue area and tumor content determined from H&E stained section of same block. Due to high amount of paraffin associated with the CNB samples, the following deparaffinization steps were performed before proceeding with the standard AVENIO Tumor Tissue DNA extraction workflow.

1. Add 800 μl xylene to each of the CNB samples, incubate 5 minutes, and vortex briefly in several intervals.

2. Add 400 μl absolute ethanol and vortex briefly. Centrifuge for 2 minutes at maximum speed (16,000 x g) and discard supernatant. Be careful not to dislodge the tissue pellet.

3. Add 1 ml absolute ethanol and vortex briefly. Centrifuge for 2 minutes at maximum speed (16,000 x g) and discard supernatant. Be careful not to dislodge the tissue pellet.

4. Blot the tube briefly onto a paper towel to get rid of ethanol residues. Dry the tissue pellet for 10 minutes at +56°C (open tubes). If required, dry for up to a maximum of 20 minutes.

For FNA samples, one slide was used. FNA cells were scraped and collected into a tube for the standard AVENIO Tumor Tissue DNA extraction workflow.

1

For Research Use Only. Not for use in diagnostic procedures.

Sequencing and Data AnalysisSequencing was conducted on a NextSeq 500 with 22 samples per sequencing run. The AVENIO Oncology Analysis Software Version 2.0.0 was used to analyze sequencing data for sequencing metrics and variant calling. RocheDefault filter set was applied to VCFs to remove germline variants by removing variants with 0.001 or greater population frequency in dbSNP, ExAC, or 1000 Genomes, and retaining variants with evidence in COSMIC or TCGA.

Results Sample Metrics and Pre-sequencing Metrics In the AVENIO Tumor Tissue Analysis Kits protocol, a normalized quality ratio (Q-ratio) is calculated and used to determine the quality of input DNA and optimal input mass into library preparation. For optimal assay performance, samples are recommended to have a Q-ratio greater than or equal to 0.04 and input DNA mass of (10/Q-ratio + 10) ng.

As shown in Table 1, 84 samples were extracted using the

modified AVENIO tumor tissue DNA extraction procedures described above. The 56 out of 84 samples (67%) that passed both Q-ratio and input mass requirements were included in the workflow. Out of the 28 samples that failed input QC, 12 samples (4 CNBs and 8 FNAs) failed the Q-ratio requirement and 16 (8 CNBs and 8 FNAs) samples passed the Q-ratio requirement but failed the input mass requirement. Given the challenge of obtaining optimal DNA from CNB and FNA samples, 10 samples (3 CNBs and 7 FNAs, marked with * and ** in Table 1) that partially met input requirements were included in the downstream workflow despite expectations that assay performance on these samples may be suboptimal. In total, 66 (50 CNBs and 16 FNAs) out of 84 tissue samples (78.5%) were processed for sequencing. Figure 1 shows the sample metrics (1a, 1b) of all 84 CNB and FNA samples and pre-sequencing metrics (1c-1f) of 66 samples processed for sequencing. Interestingly, all 6 reported metrics of CNBs turned out be significantly different from those of FNA samples (Mann–Whitney test, p-value <0.05).

Figure 1: Sample metrics and pre-sequencing metrics. The figure includes box plots showing the median, interquartile range for each metric. The whiskers extend to the smallest and largest value no further than 1.5* interquartile range from the quartiles. (a) Q-ratio, a normalized ratio to measure the quality of DNA. (b) % of recommended input mass used, recommended input was calculated based on Q-ratio using the equation 10/Q-ratio + 10. (c) Library concentration (ng/μL) before enrichment measured by Qubit. (d) Library mean size (bp) before enrichment measured by Tapestation. (e) Enriched library concentration (ng/μL) measured by Qubit. (f) Enriched library mean size (bp) measured by Tapestation.

Table 1. Sample summary

CNB FNA Total

# of samples extracted 59 25 84

# of samples passed both input requirements and included for sequencing 47 9 56

# of samples failed the Q-ratio ≥0.04 requirement 4 5(3*) 9(3*)

# of samples passed Q-ratio ≥0.04 but failed the input mass requirement 5(3**) 4(4**) 9(7**)

# of samples failed input requirements but included for sequencing 3 7 10

# of samples sequenced 50 16 66

* # of samples with 100% of recommended input mass** # of samples with >40% of recommended input mass

2

(a) Q-ratio0.60.40.20.0

(b) % of recommended input mass used

100

80

60

(c) Library concentration [ng/µL] before enrichment

50403020100

(d) Library mean size [bp] before enrichment

450400350300250

(e) Enriched library concentration [ng/µL]

10.07.55.02.50.0

(f) Enriched library mean size [bp]

360340320300280

CNB FNA

For Research Use Only. Not for use in diagnostic procedures. 3

Figure 2: Sequencing metrics. The figure includes box plots showing the median, interquartile range for each metric. The whiskers extend to the smallest and largest value no further than 1.5* interquartile range from the quartiles. (a) Number of read pairs, total number of paired-end sequencing reads (M) per sample. (b) On-target rate, the percentage of reads in the intended target region. (c) Unique depth, median read depth across the selector after removing duplicate reads. (d) Percentage of panel exon regions with ≥300× coverage, the percentage of the panel exon regions covered by greater or equal to 300 unique reads per position after removing duplicate reads. (e) Percentage of bases within 10-fold range of median depth, the percentage of positions whose unique depth fall within 10-fold of the median unique depth. (f) Error rate, the percent of non-reference bases observed over total bases, excluding variants detected at >5% AF (likely real variants).

(a) Number of read pairs [M]

18

14

10

(b) On-target rate908070605040

(c) Unique depth

3000

2000

1000

0

CNB

FNA

(d) % Panel ≥ depth 300100

75

50

25

0

(e) Uniformity

99

98

97

96

95

(f) Error rate0.20

0.15

0.10

0.05

0.00

Sequencing Metrics Key sequencing metrics of the 66 sequenced libraries are plotted in Figure 2. On-target rate, unique depth and error rate of CNB samples were significantly different from those of FNA samples (Mann–Whitney test, p-value <0.05).

Based on theoretical modeling, we determined that a unique read depth of at least 300× is a sufficient depth to robustly differentiate true variants from background noise and achieve a limit of detection (LOD) of 5% allele frequency. Therefore, in this study we defined 90% of panel exon regions with ≥300× coverage to represent sequencing success. Based on this metric, genomic

profiles were successfully generated from 55 of 56 optimal samples (98.2%) which included 46 CNB and 9 FNA samples, and from 9 of 10 sub-optimal samples (90%) which included 3 CNB and 6 FNA samples. The optimal CNB sample that failed the sequencing run did achieve expected values for all other reported sequencing metrics, but showed 86.9% of panel exon regions with unique read depth ≥300× coverage, barely missing the 90% cutoff. In contrast, the sub-optimal FNA sample (56.9% of recommended input) failed the sequencing run with 2.7% of panel exon regions with ≥300× coverage and its number of read pairs was significantly lower than others.

Variant Analysis Variant analysis was performed on the 64 samples with >90% of panel exon regions with unique read depths ≥300×. Among the 64 samples, there were 61 specimens (57 lung samples and 4 CRCs) with one or more mutations detected. The identified genomic alterations included SNVs, Indels, and CNVs (Figure 3). No fusions were detected. In total, 137 genomic alterations were identified in the 57 mutant lung samples (range 1-7 genomic alterations per sample). The most common alterations were observed in TP53 (36%), KRAS (7%) and EGFR (7%). 19 genomic alterations were identified in the 4 mutant CRC

samples (range 2-8 genomic alterations per patient). The most common alterations were observed in KRAS (26%), PIK3CA (26%) and TP53 (26%).

Even though the variant analysis summarized above excluded the sample with 86.9% of panel exon regions with ≥300× coverage, that sample had two different TP53 mutations at around 39% allele frequency, together with a MET CNV, indicating >90% of panel exon regions with unique read depth ≥300× is not required to detect variants with >5% allele frequencies.

Method Concordance Among the 64 samples successfully sequenced, there were 14 samples for which the gene status of one or more genes was tested. There were 11 samples with KRAS mutation status, 10 samples with EGFR mutation status, and 5 samples with EML4-ALK mutation status available from a tumor sample from the same resection of the same patients by PCR, NGS, FISH (fluorescence in situ hybridization) or CISH (chromogenic in situ hybridization) staining.

In all 11 KRAS cases, the AVENIO results are concordant with the reported KRAS status in the gene status testing.

In 6 out of the 11 cases, the gene status was positive, whereas in 5 out of the 11 cases, the gene status was negative. For EGFR and EML4-ALK, the AVENIO results are concordant with the reported status in the gene status testing. Both are negative in all cases. Overall, AVENIO Tumor Tissue Analysis Kits showed 100% concordance with reported KRAS, EGFR and EML4-ALK genes status by orthogonal methods in all 26 tested cases.

Figure 3: Samples with known variants SNVs, Indels and CNVs

CRC

coun

t

Lung

Aberration

50

40

30

20

10

0

TP53

KRA

SA

PCPI

K3CA

FBXW

7

TP53

KRA

SEG

FRM

ETA

PCPI

K3CA

NFE

2L2

RB1

ERBB

2FB

XW7

KEA

P1ST

K11

CDKN

2AFL

T4BR

AF

FLT3

PDCD

1LG

2PT

EN RET

FGFR

2FG

FR3

KIT

MA

P2K1

MA

P2K2

NTR

K1PD

GFR

APD

GFR

BPM

S2TS

C1

CNV

Indel

SNV

Comprehensive genomic profiling can be performed using AVENIO Tumor Tissue Analysis Kits on core needle biopsy or fine needle aspiration samples. While CNB and FNA samples tend to yield limited DNA, for most of the cases, even when sub-optimal samples were used, successful genomic profiles can be reliably obtained and the genomic alterations discovered correlated well with those identified in matched resected tumors from the same patient.

Conclusion

For Research Use Only. Not for use in diagnostic procedures.

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© 2019 Roche Sequencing Solutions, Inc. All rights reserved. MC--01823 07/2019

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