High Frequency of HLA-Class II B Cell Receptor Neoantigens in IGHV Mutated-CLL
Authors: Sabdia Muhammed B. , Burgess Melinda , Law Soi Cheng , Thurgood Lauren A , Kazakoff Stephen , Swain Fiona , Tobin Joshua W.D. , Mollee Peter , Patch Ann-Marie , Gandhi Maher K 
Published: December 2023

Introduction: Chronic lymphocytic leukemia (CLL) is the most common hematological malignancy and remains incurable. IGHV mutational status is an independent predictor of outcome with chemoimmunotherapy, however the mechanism(s) underpinning the superior prognosis of M-CLL are still to be elucidated. Clonal T cell expansions are well described in CLL. Neoantigens are immunogenic peptides unique to malignant cells, which are presented to host T cells via HLA-I/II, and if harnessed may have potential as a novel immunotherapeutic strategy. It is believed CLL has a low tumor mutational burden (TMB) due to few somatic mutations (~1/Mb) and hence few somatic neoantigens. However, traditional concepts of TMB fail to recognize that the IGH and IGκ/λ chains of the B cell receptor (BCR) also become highly mutated during B cell maturation. BCR mutations in B cell malignancies can create ‘BCR neoantigens’ (Khodadoust, Nature 2017 / Blood 2019). There is no large-scale analysis of HLA-I/II somatic and BCR neoantigens in CLL.
Methods: Pre-therapy PBMCs from 23 patients with CLL (M17:F6, median 69 yrs, M-CLL=10, U-CLL=13) were tested. Cytogenetics and patient characteristics were similar between M-CLL and U-CLL. Samples underwent paired WES and RNAseq. Somatic mutations were called by Strelka2 and somatic neoantigens by pVACseq (TESLA guidelines; Wells, Cell 2020). BCR neoantigens were called from full-length BCR sequences by pVACbind. Neoantigen transcriptome expression was confirmed in all cases.
Results: Importantly, expression of genes important for CLL antigen presentation was almost universally preserved. Furthermore, consistent with T cells controlling disease progression, central memory/effector T cells were expanded in both M-CLL and U-CLL by CIBERSORTx. Next, the somatic mutational landscape was characterized. WES revealed a median 1.06 somatic mutations/Mb, with frequency of driver mutations as expected (Knisbacher, Nature 2022). Only a median 22% of mutations/case were expressed, with expression of driver mutations only modestly increased in U-CLL vs M-CLL (1 vs 0, p=0.024), suggesting that enrichment of driver mutations in U-CLL only partially explains its inferior prognosis. Strikingly, unlike somatic mutations, all BCR mutations were expressed, with 12x &gt; BCR mutations expressed in M-CLL vs U-CLL (37 vs 3, p&lt;0.0001). Furthermore, in M-CLL expressed BCR mutations were 3x &gt; somatic mutations, whereas BCR mutations were 3x &lt; somatic mutations in U-CLL.
Neoantigens were then assessed (Figure 1). Combining somatic + BCR neoantigens, there were a median 24 neoantigens/case. Somatic neoantigens included those found in driver mutations (ATM, XPO1, SF3B1, TP53, NOTCH1). Expression of driver neoantigens were retained in sequential patient samples and new driver neoantigens also acquired. Importantly, public neoantigens (i.e., neoantigens shared across CLL patients) in TP53 and XPO1 were detected. Notably, the frequency of BCR neoantigens was higher than somatic neoantigens, with differences most marked and enriched for HLA-II (HLA-I: 5 vs 2, p&lt;0.0001 and HLA-II: 17 vs 3, p&lt;0.0001). Strikingly, there were 2x &gt; BCR neoantigens in M-CLL vs U-CLL (HLA-I: 6 vs 2, p&lt;0.001; HLA-II: 23 vs 15, p=0.032). Interestingly, the nature of BCR neoantigens between M-CLL and U-CLL was different. Consistent with M-CLL B cells undergoing somatic hypermutation, IGHV mutations strongly positively correlated with numbers of V region BCR neoantigens (R 2=0.784, p&lt;0.0001), and weakly negatively correlated with V(D)J recombination region BCR neoantigens (R 2=0.3992, p&lt;0.005). As expected, there was no correlation between the number of IGHV mutations and somatic neoantigens.
Conclusions: The reported low TMB in CLL does not reflect the true frequency of neoantigens, as it fails to account for BCR neoantigens. These contribute ~80% of the neoantigen pool and are predominantly presented by HLA-II. The majority of BCR neoantigens in M-CLL are within the V region, whereas in U-CLL, BCR neoantigens are largely restricted to the V(D)J recombination site. The known enrichment of driver mutations in U-CLL coupled with the increase of BCR neoantigens in M-CLL, are the likely mechanisms behind the different survival outcomes between M-CLL and U-CLL. The presence of public neoantigens in driver mutations opens up the possibility of off-the-shelf neoantigen specific immunotherapy and warrants further investigation.