STAT6 mutations in cHL. (A) Secondary structure of the STAT6 protein, with the mutations found in cHL cases (green circles) clustering in the DNA binding domain. The latter is enlarged below to depict individual STAT6 alleles from 6 patients each carrying multiple monoallelic STAT6 mutations. (B) Sanger-sequencing validation in 1 representative case confirms the presence of a heterozygous STAT6 mutation (D419G) in both tumor WGA-DNA duplicates, but not in matched normal WGA-DNA. (C) Digital polymerase chain reaction analysis allows the backtracking of a somatic N417Y mutation in the unamplified tumor DNA of case 14 extracted from whole-tissue sections of a lymph node biopsy (left), but not in the DNA of a peripheral blood sample analyzed as a negative control (right); the same mutation had been originally identified by WES in the WGA-DNA of microdissected tumor vs normal cells. The low variant allele frequency (0.9%) reflects the known paucity of cHL tumor cells in the involved tissues (see supplemental Table 6 for the full results of digital polymerase chain reaction validation).