Thyroid MALT lymphoma: self-harm to gain potential T-cell help

Abstract

The development of extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is driven by chronic inflammatory responses and acquired genetic changes. To investigate its genetic bases, we performed targeted sequencing of 93 genes in 131 MALT lymphomas including 76 from the thyroid. We found frequent deleterious mutations of TET2 (86%), CD274 (53%), TNFRSF14 (53%), and TNFAIP3 (30%) in thyroid MALT lymphoma. CD274 was also frequently deleted, together with mutation seen in 68% of cases. There was a significant association between CD274 mutation/deletion and TNFRSF14 mutation (p = 0.001). CD274 (PD-L1) and TNFRSF14 are ligands for the co-inhibitory receptor PD1 and BTLA on T-helper cells, respectively, their inactivation may free T-cell activities, promoting their help to malignant B-cells. In support of this, both the proportion of activated T-cells (CD4+CD69+/CD4+) within the proximity of malignant B-cells, and the level of transformed blasts were significantly higher in cases with CD274/TNFRSF14 genetic abnormalities than those without these changes. Both CD274 and TNFRSF14 genetic changes were significantly associated with Hashimoto's thyroiditis (p = 0.01, p = 0.04, respectively), and CD274 mutation/deletion additionally associated with increased erythrocyte sedimentation rate (p = 0.0001). In conclusion, CD274/TNFRSF14 inactivation in thyroid MALT lymphoma B-cells may deregulate their interaction with T-cells, promoting co-stimulations and impairing peripheral tolerance.

Fig. 1. Genetic profile of thyroid MALT lymphoma.

Data shown here include chromosome translocations associated with MALT lymphoma, and genes mutated at a frequency of ≥3%. CD274 mutation/deletion, TNFRSF14 and TET2 mutation are most frequent, often occurring together. For complete genetic data from this study, please see Supplementary Fig. S4. CNV copy number variation.

Fig. 2. Distribution and characteristics of CD274, TNFRSF14, TET2, TNFAIP3, CXCR5, CXCR3 and CCR6 mutations in thyroid MALT lymphoma.

Where possible, DNA from microdissected non-neoplastic cells was used for PCR and Sanger sequencing to exclude potential germline variants, and mutations confirmed to be somatic are indicated by symbols in red colour (Color figure online).

Fig. 3. Comparison of CD274, TNFRSF14, TET2 and TNFAIP3 mutation AAF (alternative allele frequency) in thyroid MALT lymphoma. Both TET2 and TNFRSF14 have a significantly higher mutation AAF than TNFAIP3.

TNFRSF14 also has a higher mutation AAF than CD274, although not statistically significant.

Fig. 4. Comparison of mutation burden and characteristics according to TET2 mutation status in thyroid MALT lymphoma.

TET2 mutation is excluded from the mutation calculation. A The overall mutation load is significantly higher in the cases with TET2 mutation than those without the mutation (p = 0.03). B The proportion of transition mutations is also higher in cases with TET2 mutation than those without the mutation, although not statistically significant.

Fig. 5. Multiplex immunofluorescent staining reveals increased activated T-cells in the vicinity of malignant B-cells harbouring CD274/TNFRSF14 genetic changes.

A An example of multiplex immunofluorescent staining in a case of thyroid MALT lymphoma with both CD274 and TNFRSF14 genetic changes; Diffuse tumour areas are marked, and analysed using Halo-V3.1 HighPlex FL and proximity modules. GC germinal centre. B Quantitative analysis CD4+/CD20+ cell ratio (left panel), and various CD4+ immunophenotypic subsets within 10 µm of CD20+ B-cells. The proportion of activated T-cells (CD4+CD69+/CD4+) is significantly higher in cases with CD274/TNFRSF14 genetic abnormalities than those without these changes.

Fig. 6. Correlation between CD274/TNFRSF14 genetic changes and histological features.

A Examples of grading for semi-quantification of transformed blasts in thyroid MALT lymphoma; HPF high power field. B Comparison of the extent of transformed blast, plasmacytic differentiation and follicular colonisation between cases with and without CD274/TNFRSF14 genetic changes. The extent of these histological features was scored as described in the “Method” and compared using the Wilcoxon rank sum test.

Fig. 7. Working model of molecular mechanisms underlying thyroid MALT lymphoma.

Inactivation of both CD274 (PD-L1) and TNFRSF14 in the lymphoma B-cells abolish their inhibitory regulation to T-helper cells, thus liberating T-cell function, leading to exaggerated T-cell help signals to the lymphoma B-cells.