The correlations of tumor mutational burden among single-region tissue, multi-region tissues and blood in non-small cell lung cancer

Abstract

High-level tissue tumor mutational burden (tTMB) or blood TMB (bTMB) are associated with better response of immunotherapy in non-small cell lung cancer (NSCLC) patients. However, the correlations of single-region tTMB, multi-region tTMB and bTMB remain to be determined. Moreover, whether intratumor heterogeneity (ITH) has impact on TMB should be clarified. We collected multi-region tumor tissues with matched blood from 32 operative NSCLC and evaluated single-region tTMB, multi-region tTMB and bTMB through a 1021-gene panel sequencing. TMB of > 9 mutations/Mb was classified as high. Besides, we used tTMB fold-change to evaluate the influence of the enrolled region number on tTMB. We found both of single-region tTMB and bTMB showed strong correlations with multi-region tTMB, while the former correlated better (Pearson r = 0.94, P = 2E-84; Pearson r = 0.47, P = 0.0067). It showed extremely high specificity (100%) but low sensitivity (43%) when using bTMB to define TMB-high patients, while most false-negative predictions were in early-stage patients. Compared to single region, we found significantly enhanced tTMB fold-change if taking multi-regions for consideration. However, it showed insignificant tTMB fold-change increase if the included regions' number more than three. Moreover, ITH-high patients had significantly higher tTMB fold-change compared with ITH-low patients (2.32 vs. 1.02, P = 8.879e-05). The conversion rate of tTMB level (tTMB-low to tTMB-high) was numerically higher in ITH-high group than that in ITH-low group (16.67% vs. 3.84%). In summary, single-region tTMB has stronger correlation with multi-region tTMB compared with bTMB. ITH has an impact on tTMB, especially in high-level ITH patients.

Keywords: Blood TMB; ITH; NSCLC; Tissue TMB; bTMB; tTMB.

Fig. 1

The correlations of TMB among single-region tissue, multi-region tissues and blood. a Single-region tTMB vs. multi-region tTMB & bTMB vs. multi-region tTMB. The Pearson correlations were performed between single-region tTMB and multi-region tTMB (blue, r = 0.94), and between bTMB and multi-region tTMB (red, r = 0.47). TMB reflected somatic non-synonymous SNVs, insertions, and deletions per megabase of the panel region. Single-region tTMB was defined as the number of somatic non-synonymous mutations per megabase from single region. Multi-region tTMB was calculated with non-repetitive mutations from all regions per megabase. bTMB was analyzed with tumor-derived mutations from ctDNA. b bTMB level evaluation using multi-region tTMB level as standard. The Pearson correlations between bTMB and multi-region tTMB in all patients (left), I-II stage patients (middle), and III-IV stage patients (right) were 0.47, 0.47 and 0.58, respectively. Tumor stage were evaluated following the guidelines in the International Staging System for Lung Cancer, 7th edition. The sensitivity (true positive rate: the number of high-level bTMB & high-level multi-region tTMB patients divided by the number of high-level multi-region tTMB patients) and specificity (true negative rate: the number of low-level bTMB & low-level multi-region tTMB patients divided by the number of low-level multi-region tTMB patients) were analysed using multi-region tTMB level as golden standard. TMB of > 9 mutations/Mb was classified as high, using the top quartile threshold of 2000 samples from database of Geneplus. Abbreviations: TMB, tumor mutational burden; tTMB, tissue TMB; bTMB, blood TMB; r, Pearson correlation coefficient

Fig. 2

tTMB fold-change based on multi-regions compared with single region. a For overall patients. For overall analysis, tTMB fold-change is computed by the mean tTMB of two-regions, three-regions, four-regions, five-regions, six-regions, seven-regions or eight-regions through a random iterated algorithm, then divided by that of the single region. b For different NSCLC subtypes. For subgroup analysis, tTMB fold-change is computed by the mean tTMB of two-regions or three-regions through a random iterated algorithm, then divided by that of the single region, because it shows insignificant tTMB fold-change increase if the included regions’ number more than three. Abbreviations: TMB, tumor mutational burden; tTMB, tissue TMB

Fig. 3

Impact of ITH on TMB assessment. a tTMB fold-change comparison between ITHi-high and ITHi-low patients. tTMB fold-change is computed by the mean tTMB of three-regions through a random iterated algorithm, then divided by that of the single region. b The conversion rate of tTMB level status between ITHi-high and ITHi-low patients if taking multi-region tTMB analysis. ITH index (ITHi) was calculated with specific formula displayed in Additional file 2: Supplementary Methods. ITHi ranges from 0 (lowest ITH) to 1 (highest ITH). If the tumor has less shared somatic genetic alterations after multi-region sequencing, the ITHi of this tumor will be higher. Otherwise, the ITHi of this tumor will be lower. Abbreviations: ITH, intratumor heterogeneity; TMB, tumor mutational burden; tTMB, tissue TMB