Next Generation Sequencing (NGS) of Peripheral Blood As an Alternative for NGS of Bone Marrow in the Diagnosis of Hematologic Malignancies : A Retrospective Study of Ocean County, New Jersey, USA
Authors: Shambhavi Shreya , Di Vanna Mariela , Eltoukhy Hussam 
Published: December 2024

Introduction:
Bone marrow biopsies aid in identifying hematological malignancies in patients with blood count abnormalities such as cytopenia. Although accurate, they are invasive and painful causing hesitation in patients ultimately leading to delay in diagnosis of malignancies. Next generation sequencing (NGS) is the simultaneous sequencing of thousands of DNA and RNA segments to identify mutations that can potentially lead to cancer. It can serve as an alternative screening tool to identify hematologic malignancies and can be helpful in cancer management, risk assessment and genetic counseling by providing insights into tumor clonality. [1] Jumniensuk et al [2] evaluated 163 patients who underwent NGS testing for presumed hematologic malignancy performed on peripheral blood and bone marrow within a one-year interval of each other and found that there was a 92% concordance between the two tests with a kappa coefficient of 0.794 (p&lt; 0.0001). Drawing from this study, we performed a retrospective study on our diverse patient population in Ocean County, New Jersey (NJ) to compare NGS results for paired peripheral blood (PB) and bone marrow (BM) samples for mutational analyses of hematological malignancies.
Methods:
This is a retrospective observational study performed utilizing EPIC electronic medical records for patients of oncology outpatient clinics located in Toms River, NJ and Long Branch, NJ. The study consisted of 65 patients aged between 18-88 who underwent both Peripheral blood NGS and bone marrow NGS within a timeframe of two years for workup of hematologic malignancies. Patients that were included did not have a history of bone marrow transplant and had not received treatment at the time of the tests, such as chemotherapy, radiation or surgery. Nucleic acid was isolated from peripheral blood smear specimens and NGS library preparation was done through anchored multiplex PCR. Alternatively, Amplicon based next generation sequencing was done on the bone marrow specimens. The correlation between NGS analyses of paired bone marrow and peripheral blood samples was calculated using kappa coefficient correlation analysis. The correlation between variable allele frequency (VAFs) of paired PB/BM samples was calculated using Pearson correlation analysis.
Results:
Overall, 181 variants in 31 different genes were detected in 130 samples. The mean (SD) [min-max] number of mutations evident in the bone marrow was 1.57 (1.61) [0-6] and in the peripheral blood the mean (SD) [ min-max] number of mutations was 1.45 (1.43) [0-5]. High concordance 95.3% (62/65), strong correlation (kappa coefficient 0.90, p&lt;0.05) was found between NGS analyses of paired samples of peripheral blood and bone marrow with sensitivity and specificity being 98% and 92% respectively. The study showed 38 completely concordant cases, 24 partially concordant cases and 3 discordant cases. Variable allele frequency (VAFs) between NGS analyses of paired PB and BM samples were found to be strongly correlated in the overall sample (r=0.85, p&lt;0.05). A total of 7/181 (3.8%) detected mutations were discordant, 6 of which had a VAF&lt;5.2%. Among patients of the white race, the concordance between the paired BM and PB samples was found to be 93.6%, with the kappa coefficient being 0.87 and 100% among Non-White population which included patients of Hispanic, Asian and African American origin. It is important to note that only 28% of our sample identified as Non-White.
Conclusion:
The study demonstrated a high degree of concordance and great agreement between NGS analyses of paired PB/BM samples. Our study concludes that NGS peripheral blood can be reliably used as a noninvasive alternative for bone marrow NGS for identification of carcinogenic mutations and molecular classification of hematological neoplasms without loss of sensitivity and specificity.
References: Qin D. Next-generation sequencing and its clinical application. Cancer Biol Med. 2019;16(1):4-10. doi:10.20892/j.issn.2095-3941.2018.0055Jumniensuk C, Nobori A, Lee T, Senaratne TN, Rao D, Pullarkat S. Concordance of Peripheral Blood and Bone Marrow Next-Generation Sequencing in Hematologic Neoplasms. Adv Hematol. 2022;2022:8091746. Published 2022 Mar 26. doi:10.1155/2022/8091746