High Frequency of Subclonal Myeloid Mutations Can be Found in Large Granular Lymphocyte Leukemia

Large granular lymphocyte (LGL) leukemia is a clonal lymphoproliferation of cytotoxic T-lymphocytes often associated with immune cytopenias. Somatic mutations in STAT3 were found in a proportion of LGL and provide a good diagnostic marker for positive cases but a large proportion of LGL cases is negative. More polyclonal CTL reactions directed against hematopoietic stem and progenitor cells operate in aplastic anemia but have been also postulated as a pathogenetic factor leading to cytopenias in some cases of myelodysplastic syndromes (MDS). Increased CTL activation and LGL can be occasionally seen in MDS and previously, concomitant LGL and MDS have been described. According to one theory behind LGL pathogenesis, proliferation is driven by a more or less specific tumor surveillance reaction. Moreover, asymptomatic clonal hematopoiesis, T cell clonopathy of unclear significance as well as LGL show an overlapping demographic.

By applying a rational screening algorithm for LGL in MDS (to identify treatable causes of immune cytopenia) we have identified patients with concomitant MDS and T-LGL and determined their somatic molecular profile. We have hypothesized that some of the T-LGL cases may be associated with the presence of clonal somatic mutations seen in MDS, consistent with subclinical MDS or clonal hematopoiesis of indeterminate potential (CHIP). We stipulated that LGL may indeed correspond to more or less successful tumor surveillance reaction and thus myeloid mutations would be more commonly seen in LGL than expect based on age. Consequently, according to the reverse principle we have screened patients with LGL for the presence of clonal myeloid mutations.

We have studied 225 LGL patients; among them by traditional pathomorphologic criteria we have identified 9 (3%) patients with concomitant MDS; 1/9 had STAT3^D661V, 8/9 patients had chromosomal abnormalities, including del(20q) (n=1), del(5q) (n=2), -X (n=1), -7 (n=3) and complex karyotype in 1 patient. When "pure" LGL patients were assessed using deep NGS we found STAT3 mutations in 27% of our LGL cohort including most common D661V/Y followed by Y640F but also less frequent hits such as N647I, S614R, K658*/H/N mainly mapped in the SH2 domain (584-674) of the protein. Conversely, the analysis of 472 MDS did not identify any STAT3 mutations.

A subset of LGL patients without evidence for the morphologic or cytogenetic presence of MDS (n=66; excluding the cases with concomitant MDS) were then selected for targeted NGS with a panel containing 65 genes recurrently found in myeloid neoplasms (MDS panel). Excluding STAT3 mutations, hits in myeloid cells were found in 36/66 (55%) of patients with 12 myeloid mutations in STAT3^MT and 24 mutations in STAT3^WT. For those with clonal events the average number of mutations was 2.3 and average VAF was 17-57%. The most common mutated genes included: KMT2D (6/66; 9%), KDM6A/B (5/66; 8%), TET2 and (3/66; 5% each), and ASXL1 (2/66; 3%) SF3B1, CUX1, GNAS, IDH1, NPM1 among others (e.g., ATR, BARD1, BRIP1, CDKN2A, SETD2; N=17 ) .

In conclusion, somatic mutations in common "myeloid" genes can be found in patients with LGL without morphologic presence of MDS. The much higher than expected rate detection in LGL (median age of 63 yrs.) when compared to the elderly patients with clonal hematopoiesis (NEJM 2014, Jaiswal et.al) may lead to a stipulation that the presence of aberrant myeloid clones may be sensed by LGL and thus some of LGL occurring in older patients may reflect an abortive tumor surveillance reaction. Targeted gene sequencing has helped identify a spectrum of MDS-related subclonal myeloid mutations in LGL and thereby further our understanding of the pathogenesis of LGL.