Utilization of Non-Invasive Sample Sources for Identification of Somatic and Germline Mutations in Hematologic Malignancies and Clonal Hematopoiesis
Authors: Soyfer Eli M , Nguyen Hellen , Bhardwaj Simran , Lai Hew Yeng , Haghighi Nahid , Fleischman Angela 
Published: November 2021

Abstract
Introduction
A matched "tumor" and germline sample is required to unambiguously identify somatically acquired mutations in the blood or bone marrow of patients with hematologic malignancies or clonal hematopoiesis, particularly when variants of unknown significance (VUS) with a variant allele frequency (VAF) approximating 50% are encountered. Our goal was to identify sample sources for matched blood and germline samples to fit the following criteria: 1) non-invasive, 2) amenable to home collection by participants, and 3) stable at room temperature for an extended period. We hypothesized that saliva, comprised of mainly white blood cells as the DNA source, is a viable alternative to blood for identifying somatic hematopoietic mutations and that nail clippings provide adequate DNA devoid of contamination from blood cells to serve as a germline sample. To test this, a targeted NGS myeloid panel was performed on concurrently collected blood, saliva, and nail samples from patients with myeloproliferative neoplasms (MPN) or other clonal disorders.
Methods
Peripheral blood was collected in EDTA tubes, aliquoted into microcentrifuge tubes, and stored at -80°C until use. Saliva was collected using a DNA/RNA shield saliva collection kit (Zymo) and stored at -80°C until use. Fingernail or toenails were clipped into a plastic bag and stored at room temperature until use. For isolation of DNA from blood the DSP DNA Blood mini kit (QIAGEN) was used. For saliva and nails, the QIAmp DNA Investigator kit (QIAGEN) was used. After DNA quantification targeted NGS libraries were prepared from 100ng of DNA per sample using the ArcherDX VariantPlex Myeloid (SK0123) workflow. The resulting libraries were sequenced on an Illumina NextSeq500 instrument using v2 chemistry (Illumina, San Diego, CA), obtaining at least 4 millionreads per sample. The FASTQ data files were analyzed on ArcherDX Suite Analysis software (v.5.1.7) to identify SNPs, Indels, structural rearrangements, and Copy Number Variations.
Results
First, to test if nail clippings are a feasible source of germline DNA, we performed a targeted NGS myeloid panel on paired blood and nail from a patient with Polycythemia Vera (Patient #1). Clippings from approximately five nails was sufficient to yield 100ng of DNA. JAK2 V617F was detected at a low VAF in his nail sample (4% in nail vs 23% in blood), suggesting the presence of blood contamination. On subsequent samples, nail clippings were rinsed with water prior to DNA purification, and no MPN driver mutations were detected in washed nails (Patients #2-5). However, since processing of nails for DNA purification is time consuming and laborious, and the DNA yield from nails is quite limited, we are investigating alternative non-invasive sources for germline samples including nasal swabs.
Sequencing of matched blood and saliva was performed from patients with all three MPN driver mutations, and in a patient with Clonal Cytopenia of Undetermined Significance (CCUS). All somatic mutations identified in the blood were also seen in the saliva, almost all had identical VAFs from both sample sources, even with VAFs as low as 3%.
Conclusion
Nail clippings are a feasible source for germline DNA in patients with hematologic malignancies and clonal hematopoiesis. Saliva is equivalent to peripheral blood for identifying somatic mutations in hematopoietic cells. These methods of sample collection are non-invasive, amenable to in home collection, and are temperature stable. Collection of saliva and nails could be easily utilized for remotely administered studies by mailing collection kits to participants, thus avoiding the cost and labor of a blood draw.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.