For Research Use Only. Not for use in diagnostic procedures.
The KAPA HyperCap Design Share (DS) non-Hodgkin lymphoma (NHL) panel enables highly sensitive detection and longitudinal analysis of ctDNA in blood-derived NHL samples for research use purposes. Existing methods such as the radiological and nuclear imaging methods are unable to provide key information on clonal evolution and minimal residual disease (MRD).1
Cell-free circulating tumor DNA (cfDNA/ctDNA), typically assessed using targeted deep sequencing, has emerged as an important non-invasive and highly sensitive biomarker in the monitoring of NHL patient status.2,3 To unlock the full potential of ctDNA as a biomarker more research needs to be done to better understand where it can make the most impact. Now clinical researchers have the flexibility to develop their in-house workflows using the KAPA HyperCap DS NHL panel along with the robust KAPA HyperCap workflows5 and customizable bioinformatics.6
The KAPA HyperCap Design Share NHL Panel is a research solution that covers single nucleotide variants (SNVs) in coding and/or untranslated regions of 383 genes, plus additional intergenic regions for a total capture size of 341 Kb. These genomic regions are enriched in genomic alterations associated with NHL. Used in combination with the KAPA HyperCap workflow5 and open-source KAPA bioinformatics6 analysis for longitudinal detection of ctDNA, it offers a robust, streamlined, and fully integrated solution for highly sensitive detection and longitudinal study of NHL-associated SNVs in blood-derived samples.
Figure 1. KAPA HyperCap DS NHL panel met key sequencing performance criteria and exhibited high sequencing efficiency as exhibited by the unique sequencing depth per sample.
In this study, commercial reference samples were used to prepare “contrived” samples— with known variants at allele frequencies (AFs) ranging from 0% to 5%—that mimic NHL samples. Next-generation sequencing (NGS) libraries were prepared using the KAPA HyperPrep Kit and KAPA HyperCap cfDNA Workflow v1.1 or the KAPA HyperPlus Kit and KAPA Hypercap Workflow v3.4. Libraries were enriched by hybridization to the KAPA HyperCap DS NHL Panel. Sequencing was performed on an Illumina® NextSeqTM 500/550 instrument using standard protocols. Data analysis was performed using open source bioinformatic tools.6 The KAPA HyperCap DS NHL panel sequencing metrics met expectations for a 8-sample run on Illumina® NextSeqTM 500/550 flow-cell with an average number of reads > 88M per sample. After unique molecular identifier (UMI) deduplication, the median number of reads returned for Complete Mutation Mix (Seraseq®) libraries was 44 M, compared to the median of 33 M reads for Pan-cancer Reference Standard libraries. This translated to a median coverage depth of 6100X and 5000X, respectively.
Figure 2. High sensitivity was exhibited across samples with 0.5%, 1% and 5% AFs
Each blue dot represents a replicate. SNV calling sensitivity was demonstrated by using a blended sample of gDNA NA24631 (98%) & NA24149 (2%), targeting 1% AF, with characterized variants covered by the KAPA HyperCap DS NHL Panel to assess variant calling performance for the germline workflow (DNA blend 1%: 10 true positive (TP); 8 replicates) and pre-fragmented reference cfDNA samples with known variants at specific AF covered by the KAPA HyperCap DS NHL Panel to assess somatic variant calling performance for the plasma cfDNA workflowKAPA HyperCap DS NHL Panel (Seraseq® ctDNA complete mix AF 0.5% and AF 5% from SeraCare: 3 SNVs, 2 replicates each; cfDNA Pan-cancer Reference Standard AF 0.5% and 5% from Twist: 12 SNVs, 4 replicates each). For the germline workflow, 10 out of 10 (100%) TP were observed in all gDNA replicates. For the plasma cfDNA workflow, all expected SNVs were found in all cfDNA replicates that were investigated.
*High SNV calling specificity demonstrated by 0.06 errors every 10 Kb of the panel (as calculated by a set of 23 healthy donor samples). In the gDNA NA24149/NA24631 blend, 53 out of 53 true negative (TN) were observed in all eight replicates.
Sample | n_mutations | total alt reads | p-value | decision |
---|---|---|---|---|
AF 0.5% |
9 |
199 |
0.0001 | 100% |
AF 0.1% |
9 |
43 |
0.0001 | 100% |
AF 0.05% |
9 |
25 |
0.0001 | 100% |
AF 0.01% |
9 |
11 | 0.0001 - 0.2858 | 83% |
WT (AF 0%) |
9 | 8 | 0.0001 - 0.2858 | 100% |
Table 1. Summary of longitudinal mutation analysis results for contrived cfDNA Pan-cancer reference standard samples
Data generated from samples were subsequently processed using the three-stage KAPA bioinformatics analysis for longitudinal detection of ctDNA to demonstrate the use of the KAPA HyperCap DS NHL Panel for longitudinal analysis of NHL-associated variants in circulating tumor DNA. Reporter variants were successfully detected in all contrived TN (subsequent samples after baseline) Pan-cancer samples. The Monte Carlo p-value8,9 threshold for ctDNA positivity in simulated longitudinal samples was set at 0.003 since this was the lowest value observed in the wild type sample. The number of reporter variants with non-zero supporting reads, as well as the total number of supporting alt reads, drops as the expected AF % decreases from 0.5% to 0.01%. Mutation positivity was accurately called in all replicates of the AF 0.5%, AF 0.1%, and AF 0.05% samples (Monte Carlo p-values <0.003). For the AF 0.01% sample, mutation positivity was accurately called in five out of six replicates. All replicates of the wild type sample were called negative.
The KAPA HyperCap DS NHL Panel targets 383 genes with complete or partial coverage, plus additional intergenic regions for a total capture size of 341 Kb.
References
Roschewski M, Rossi D, Kurtz DM, et al. Circulating Tumor DNA in Lymphoma: Principles and Future Directions. Blood Cancer Discov. 2022 Jan;3(1):5-15. doi: 10.1158/2643-3230.BCD-21-0029.
Scherer F, Kurtz, DM, Newman AM, et al. Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA. Sci Transl Med 2016;8(364),364ra155. doi: 10.1126/scitranslmed.aai8545.
Fernández-Miranda I, Pedrosa L, Llanos M, et al. Monitoring of Circulating Tumor DNA Predicts Response to Treatment and Early Progression in Follicular Lymphoma: Results of a Prospective Pilot Study. Clin Cancer Res 2023;29(209–220). doi: 10.1158/1078- 0432.CCR-22-1654.
Saif MW, Tzannou I, Makrilia N, Syrigos K. Role and cost effectiveness of PET/CT in management of patients with cancer. Yale J Biol Med. 2010 Jun;83(2):53-65. PMID: 20589185; PMCID: PMC2892773.
Bermejo C, Agarwal P, Chien R et al. The KAPA HyperCap Design Share NHL Panel enables highly sensitive, longitudinal detection of non-Hodgkin lymphoma circulating tumor DNA. Roche white paper. MC --11981.
Chien, R. KAPA bioinformatics analysis for longitudinal detection of circulating tumor DNA. Roche white paper. MC – 12095.
Herrera et al. Risk Profiling of Patients with Previously Untreated Diffuse Large B-Cell Lymphoma (DLBCL) By Measuring Circulating Tumor DNA (ctDNA): Results from the POLARIX Study. Blood 2022; 140 (supplement 1): 1297-1300. https://pubmed.ncbi.nlm.nih.gov/35086141/
Newman AM, Lovejoy AF, Klass DM, et al. Integrated digital error suppression for improved detection of circulating tumor DNA. Nature Biotechnol 2016;34(547–555). doi: 10.1038/nbt.3520.
Alkodsi A, Meriranta L, Pasenen A, Sirpa Leppä. ctDNAtools: An R package to work with sequencing data of circulating tumor DNA. bioRxiv 2020.01.27.912790. doi: 10.1101/2020.01.27.912790.
The KAPA HyperCap DS NHL panel is available in 24 and 96 sample sizes. For ease of ordering* there are 4 virtual kits (VKs) available that include the reagent kits that will be needed to build an in-house research workflow based on the Longitudinal detection of non-Hodgkin lymphoma ctDNA white paper.1 Please contact your local Roche sales representative to order these reagent kits to get started with NHL ctDNA monitoring research in your laboratory.
The KAPA HyperCap DS NHL panel is available in 24 and 96 sample sizes. For ease of ordering* there are 4 virtual kits (VKs) available that include the reagent kits that will be needed to build an in-house research workflow based on the Longitudinal detection of non-Hodgkin lymphoma ctDNA white paper.1 Please contact your local Roche sales representative to order these reagent kits to get started with NHL ctDNA monitoring research in your laboratory.
Material Number # | Description | Samples | Workflow and Comments |
---|---|---|---|
10040084001 |
KAPA HyperCap DS NHL cfDNA 24 samples VK |
24 |
Plasma cfDNA |
10040076001 |
KAPA HyperCap DS NHL cfDNA 96 samples VK |
96 |
Plasma cfDNA |
10040068001 |
KAPA HyperCap DS NHL gDNA 24 samples VK |
24 |
gDNA (plasma-depleted whole blood) |
10040092001 |
KAPA HyperCap DS NHL gDNA 96 samples VK |
96 | gDNA (plasma-depleted whole blood) |
09052593001 |
KAPA HyperChoice MAX 3Mb T1, 24rxn |
24 |
IRN: 1000028225 |
09052615001 |
KAPA HyperChoice MAX 3Mb T1, 96rxn |
96 |
IRN: 1000028225 |
Varies |
KAPA HyperChoice MAX 3Mb T1 |
Varies |
IRN: 1000028225, larger reaction pack sizes available-reach out to your local Roche sales representative |
References
Bermejo C, Agarwal P, Chien R et al. The KAPA HyperCap Design Share NHL Panel enables highly sensitive, longitudinal detection of non-Hodgkin lymphoma circulating tumor DNA. Roche white paper. MC --11981.