Myeloid NGS Analyses of Paired Samples from Bone Marrow and Peripheral Blood Yield Concordant Results: A Prospective Cohort Analysis of the AGMT Study Group

Abstract

Background: Next generation sequencing (NGS) has become indispensable for diagnosis, risk stratification, prognostication, and monitoring of response in patients with myeloid neoplasias. Guidelines require bone marrow evaluations for the above, which are often not performed outside of clinical trials, indicating a need for surrogate samples. Methods: Myeloid NGS analyses (40 genes and 29 fusion drivers) of 240 consecutive, non-selected, prospectively collected, paired bone marrow/peripheral blood samples were compared. Findings: Very strong correlation (r = 0.91, p < 0.0001), high concordance (99.6%), sensitivity (98.8%), specificity (99.9%), positive predictive value (99.8%), and negative predictive value (99.6%) between NGS analyses of paired samples was observed. A total of 9/1321 (0.68%) detected mutations were discordant, 8 of which had a variant allele frequency (VAF) ≤ 3.7%. VAFs between peripheral blood and bone marrow samples were very strongly correlated in the total cohort (r = 0.93, p = 0.0001) and in subgroups without circulating blasts (r = 0.92, p < 0.0001) or with neutropenia (r = 0.88, p < 0.0001). There was a weak correlation between the VAF of a detected mutation and the blast count in either the peripheral blood (r = 0.19) or the bone marrow (r = 0.11). Interpretation: Peripheral blood samples can be used to molecularly classify and monitor myeloid neoplasms via NGS without loss of sensitivity/specificity, even in the absence of circulating blasts or in neutropenic patients.

Keywords: acute myeloid leukemia (AML); bone marrow; concordance; diagnosis; myelodysplastic syndromes/neoplasms (MDS); myeloid neoplasias; next generation sequencing (NGS); peripheral blood; prognosis.

Figure 2

Occurrence of mutations by gene and by sample type for all samples (n = 150 patients, n = 191 sample pairs ^1,2, n = 1299 mutations detected ^3,4). The X-axis represents the percentage of samples in which the respective mutation was found. (¹ Includes 53 serial sample pairs from 30 patients. ² Total sample pairs (n = 240), excluding sample pairs without mutations (n = 46) and excluding sample pairs in which only fusion genes (and no other mutations) were found (n = 3). ³ Mutations occurring in genes in <2% of the total cohort were not included in the graph and included the following genes for bone marrow vs. peripheral blood, respectively: MYD88 1.7% vs. 1.7%; ETV6 1.7% vs. 1.7%; CEBPA 1.7% vs. 1.7%; PTPN11 1.3% vs. 1.3%; IKZF 1.3% vs. 1.3%; HRAS 1.3% vs. 1.3%; BRAF 0.8% vs. 0.8%; WT1 0.8% vs. 0.8%; KIT 0.8% vs. 0.8%; and RB1 0.4% vs. 0.0%. ⁴ If a sample had more than one mutation in the same gene, this was only counted one time).

Figure 4

Scatterplot of the variant allele frequency (VAF) of the mutated genes in bone marrow and peripheral blood for patient samples with the following peripheral blood parameters on the day of bone marrow sampling. (A) Peripheral blast percentage = 0% (n = 165). (B) Peripheral blast percentage ≥ 1% (n = 74). ¹ (C) Absolute neutrophil count < 1.0 G/L (n = 66). (D) Absolute neutrophil count ≥ 1.0 G/L (n = 174). The black line is the bisecting line showing a perfect linear regression with slope = 1 and intercept = 0. The red dashed line is the regression line, indicating the correlation between BM_VAF and PB_VAF of each paired sample. (¹ The peripheral blood blast percentage was missing for one patient).

Figure 1

Numbers of patients, samples and mutations. * Total reported molecular results (n = 1415) consist of: 1321 pathogenic variants (8 fragment analysis, 1313 NGS); 94 samples with no mutations.

Figure 3

Scatterplot of the variant allele frequency (VAF) of the mutated genes in bone marrow and peripheral blood. The black line is the bisecting line showing a perfect linear regression with slope = 1 and intercept = 0. The red dashed line is the regression line, indicating the correlation between BM_VAF and PB_VAF of each paired sample.

Figure 5

Scatterplots of the “variant allele frequency (VAF) of a mutation” vs. “blast percentage”. (A) “BM_VAF” vs. “percentage of bone marrow blasts”. (B) “PB_VAF” vs. “percentage of peripheral blood blasts”. The black line is the bisecting line showing a perfect linear regression with slope = 1 and intercept = 0. The red dashed line is the regression line, indicating the correlation between BM_VAF and PB_VAF of each paired sample.

Figure 6

Scatterplot of the variant allele frequency (VAF): difference against the mean. ¹ The X−axis shows the mean of the BM_VAF and the PB_VAF. The Y−axis displays the difference between the BM_VAF and the PB_VAF. The central red dashed line represents the mean of the difference, ($\stackrel{-}{\mathrm{d}}$ = 2.92), and the outer red dashed lines correspond to $\stackrel{-}{\mathrm{d}}\pm 2\stackrel{-}{\mathsf{\sigma }}$, where $\stackrel{-}{\mathsf{\sigma }}$ represents the standard deviation of the difference ($\stackrel{-}{\mathsf{\sigma }}$ = 7.71). As discussed in [55], the region between these two outer lines is referred to as the “limits of agreement” and it visualizes the difference between the two methods of measurement. (¹ Analyzed according to Bland et al., Lancet 1986 [55]).