common clinical manifestations of large granular lymphocyte leukemia ( lgl leukemia ) include cytopenias , recurrent infections , splenomegaly , and autoimmune disorders . diagnosis can be difficult , and is usually based on clinical presentation , persistent peripheral lgl lymphocytosis , and morphologic / immunophenotypic analysis . for t - lgl leukemia , a t - cell receptor ( tcr ) gene rearrangement study is performed to confirm the presence of the monoclonal t - cell population . an abnormal killer - cell ig - like inhibitory receptor ( kir ) expression pattern has been reported in some of the nk - lgl leukemia patients , although this is not routinely employed . chronic exposure to antigen or virus has been postulated to be the initial trigger in lgl leukemia pathogenesis . recent studies demonstrated that jak / stat pathway deregulation may promote clonal expansion of the activated lgl population . to further understand lgl leukemia pathogenesis the cdr3 sequences , tcrv profiles , stat3 mutation profile and the underlying signaling pathway were examined throughout the disease . the institutional review board of hershey medical center , pennsylvania state university approved this study . deep sequencing , the tcr cdr3 regions were amplified and sequenced by adaptive biotechnologies corp ( seattle , wa ) using the immunoseq assay on cdnas . real - time quantitative pcr ( qrt - pcr ) was employed to examine the tcrv profile , using primers and methods described previously , except that sybrgreen ( life technologies ) was used and target specificity was confirmed within the imgt primer database . deep sequencing results for one clone were validated using pcr - based cdr3 sequencing with primers designed from the imgt primer database ( tbv9 , 5-cac tct gaa cta aac ctg a-3 ; cb1a , 5-ggg tgt ggg aga tcc tg(c)-3 ) . conventional sanger stat3 mutation testing was carried out as previously described by koskela et al . . primer pairs were designed to cover exons 2021 encoding the stat3 src homology 2 ( sh2 ) domain . droplet digital pcr ( ddpcr ) assays were designed to detect individual mutations d661y , y640f and s614r . assays were designed to the following context sequences : d661y ggatattggtagcat(a / c)catgatcttatagcc , y640f agctgctgctttgtg(a / t)atggttccacggact , and s614r gacgcctccttcttt(t / g)ctgctttcactgaat . assays were performed in duplicate on the qx200 droplet reader and generator ( bio - rad , hercules , ca ) . western immunoblotting utilized antibodies to stat3 ( # 9139 ) , py705-stat3 ( # 9131 ) , mcl-1 ( # 5453 ) and gapdh ( # 2118 ) ( cell signaling , danvers ma ) a 77-year - old male presented in 2002 with mild macrocytic anemia and splenomegaly . based on the peripheral blood morphology , bone marrow biopsy , flow cytometry and positive tcr gene rearrangement assay , cd3+/cd8 + t - lgl leukemia was diagnosed . he did not require any therapy other than routine follow - up , which showed stable blood counts ( table 1a ) . in 2011 , his follow - up cbc revealed worsening anemia and moderate neutropenia with increased lymphocytosis . repeated flow cytometry showed a marked increase in the cd3/cd16+/cd56 population that represents the nk - cell phenotype , with a smaller cd3+/cd8 + t - cell population . these results suggested that his leukemia had evolved to nk - predominant disease with a residual of previous t - lgl leukemia ( table 1a ) . despite the progression of his leukemia , he has remained asymptomatic and clinically stable to date , and no treatment has been required . antigen exposure has been postulated as the initial stimulus for ctl expansion in t - lgl leukemia pathogenesis . we employed qrt - pcr to study the dynamic change of tcrv profile using primers designed to amplify each of the human tcrv genes ( hbv ) . as shown in table 1a , shortly after his initial diagnosis there was a predominant expression in the tcrv9 region with smaller expansions in the tcrv3 and tcrv21 regions . tcrv3 and tcrv21 usages persisted and later became predominant , while tcrv9 expression was lost , revealing a drift in tcrv usage that correlated with the t - lgl to nk - lgl phenotypic switch . cdr3 is a hypervariable region on the tcr that is critical for antigen recognition and clonal t - cell expansion . sequencing of this region in t - lgl leukemia was recently enabled by utilizing deep sequencing technology . three cdr3 sequences were detected at very high frequencies in the diagnostic t - lgl leukemia sample of 2003 ( table 1b ) . all three clones persisted throughout the disease ; however , the clone represented by the cdr3 sequence casstgdnqpqhf , which had the highest copy number initially , was significantly decreased to an extremely low copy number when the nk - lgl clone emerged . meanwhile , the other two clones became more dominant as the disease evolved , similar to oligoclonal tcrv distribution depicted in table 1a . the cdr3 sequences identified in this case appeared to be unique compared to the sequences reported in other t - lgl leukemia studies . clonal drift was previously reported in one third of t - lgl leukemia cases , although it is not strongly associated with clinical features . the worsening of cytopenias , such as this patients anemia at later dates , implies an important role of clonal drift in disease evolvement . therefore , long - term immunophenotypic monitoring may provide clinical relevance and guide medical management . it has been reported that 4070% of t - lgl and 30% of nk - lgl leukemia patients carry activating stat3 mutations . to investigate whether a deregulated stat3 pathway was associated with the patients disease progression , we searched for mutations in the stat3 sh2 domain before and after the t - lgl to nk - lgl phenotype switch . a stat3 s614r mutation was identified by sanger sequencing later in the illness in the nk - cells , but not the t - cells ( fig . 1a , data not shown for early stage ) . an elevated phosphorylated stat3 level and an increase in its transcriptional target mcl-1 1b ) , consistent with enhanced transcriptional activity of the mutant protein . in order to determine if the s614r - mutated clone had been present all along , we tested unsorted genomic dna samples from 2003 and 2011 for s614r and for two common mutations y640f and d661y by ddpcr . both samples contained approximately 35,000 copies of the stat3 locus , yielding approximately 9000 positive droplets for the wild - type allele . 1c ) exceeded the 12 positive droplets found in similar amounts of normal dna by at least 10-fold ( not shown ) . s614r mutations were not detected in the earlier sample ( no copies mutated in a background of roughly 35,000 wild - type copies ) . small but non - negligible amounts of d661y and y640f mutations suggested the presence of 8.52% and 0.68% clones , respectively , assuming heterozygous mutation . these contracted to roughly 0.1% ( 1:500 cells ) in the follow - up sample , shadowing the decrease in the t cell compartment . in contrast , s614r dominated in the follow - up specimen with a mutation rate of 33.3% and a clone size of roughly 67% , which is identical to the cd3/cd16 + population reported in table 1a . therefore , this patient developed a dominant nk - lgl clone harboring the s614r mutation , while a population of cells with a strongly - activating y640f mutation remained a minority clone . this clearly suggests that the capacity of particular stat3 mutations to drive clonal dominance can not be predicted a priori , rather it is likely to be dependent upon the genetic context of these mutations such as co - existent activating mutations or loss of suppressive checkpoint mechanisms . our findings here not only complement the known mutational mechanism for nk - lgl leukemogenesis , but also suggest that t and nk - lgl leukemia can result from the same underlying pathological process , with mutations in stat3 able to support the expansion of either cell type . in 2009 , the world health organization recognized a provisional category of chronic lymphoproliferative disorder of nk cells to distinguish it from t - cell lgl leukemia as well as aggressive nk - lgl leukemia . the clinical and molecular data presented here suggests that this distinction amongst the chronic lgl diseases may need to be reconsidered . a 77-year - old male presented in 2002 with mild macrocytic anemia and splenomegaly . based on the peripheral blood morphology , bone marrow biopsy , flow cytometry and positive tcr gene rearrangement assay , cd3+/cd8 + t - lgl leukemia was diagnosed . he did not require any therapy other than routine follow - up , which showed stable blood counts ( table 1a ) . in 2011 , his follow - up cbc revealed worsening anemia and moderate neutropenia with increased lymphocytosis . repeated flow cytometry showed a marked increase in the cd3/cd16+/cd56 population that represents the nk - cell phenotype , with a smaller cd3+/cd8 + t - cell population . these results suggested that his leukemia had evolved to nk - predominant disease with a residual of previous t - lgl leukemia ( table 1a ) . despite the progression of his leukemia , he has remained asymptomatic and clinically stable to date , and no treatment has been required . antigen exposure has been postulated as the initial stimulus for ctl expansion in t - lgl leukemia pathogenesis . we employed qrt - pcr to study the dynamic change of tcrv profile using primers designed to amplify each of the human tcrv genes ( hbv ) . as shown in table 1a , shortly after his initial diagnosis there was a predominant expression in the tcrv9 region with smaller expansions in the tcrv3 and tcrv21 regions . tcrv3 and tcrv21 usages persisted and later became predominant , while tcrv9 expression was lost , revealing a drift in tcrv usage that correlated with the t - lgl to nk - lgl phenotypic switch . cdr3 is a hypervariable region on the tcr that is critical for antigen recognition and clonal t - cell expansion . sequencing of this region in t - lgl leukemia was recently enabled by utilizing deep sequencing technology . three cdr3 sequences were detected at very high frequencies in the diagnostic t - lgl leukemia sample of 2003 ( table 1b ) . all three clones persisted throughout the disease ; however , the clone represented by the cdr3 sequence casstgdnqpqhf , which had the highest copy number initially , was significantly decreased to an extremely low copy number when the nk - lgl clone emerged . meanwhile , the other two clones became more dominant as the disease evolved , similar to oligoclonal tcrv distribution depicted in table 1a . the cdr3 sequences identified in this case appeared to be unique compared to the sequences reported in other t - lgl leukemia studies . clonal drift was previously reported in one third of t - lgl leukemia cases , although it is not strongly associated with clinical features . the worsening of cytopenias , such as this patients anemia at later dates , implies an important role of clonal drift in disease evolvement . such drift may precede changes in the disease course . therefore , long - term immunophenotypic monitoring may provide clinical relevance and guide medical management . it has been reported that 4070% of t - lgl and 30% of nk - lgl leukemia patients carry activating stat3 mutations . to investigate whether a deregulated stat3 pathway was associated with the patients disease progression , we searched for mutations in the stat3 sh2 domain before and after the t - lgl to nk - lgl phenotype switch . a stat3 s614r mutation was identified by sanger sequencing later in the illness in the nk - cells , but not the t - cells ( fig . 1a , data not shown for early stage ) . an elevated phosphorylated stat3 level and an increase in its transcriptional target mcl-1 1b ) , consistent with enhanced transcriptional activity of the mutant protein . in order to determine if the s614r - mutated clone had been present all along , we tested unsorted genomic dna samples from 2003 and 2011 for s614r and for two common mutations y640f and d661y by ddpcr . both samples contained approximately 35,000 copies of the stat3 locus , yielding approximately 9000 positive droplets for the wild - type allele . all 1c ) exceeded the 12 positive droplets found in similar amounts of normal dna by at least 10-fold ( not shown ) . s614r mutations were not detected in the earlier sample ( no copies mutated in a background of roughly 35,000 wild - type copies ) . small but non - negligible amounts of d661y and y640f mutations suggested the presence of 8.52% and 0.68% clones , respectively , assuming heterozygous mutation . these contracted to roughly 0.1% ( 1:500 cells ) in the follow - up sample , shadowing the decrease in the t cell compartment . in contrast , s614r dominated in the follow - up specimen with a mutation rate of 33.3% and a clone size of roughly 67% , which is identical to the cd3/cd16 + population reported in table 1a . therefore , this patient developed a dominant nk - lgl clone harboring the s614r mutation , while a population of cells with a strongly - activating y640f mutation remained a minority clone . this clearly suggests that the capacity of particular stat3 mutations to drive clonal dominance can not be predicted a priori , rather it is likely to be dependent upon the genetic context of these mutations such as co - existent activating mutations or loss of suppressive checkpoint mechanisms . our findings here not only complement the known mutational mechanism for nk - lgl leukemogenesis , but also suggest that t and nk - lgl leukemia can result from the same underlying pathological process , with mutations in stat3 able to support the expansion of either cell type . in 2009 , the world health organization recognized a provisional category of chronic lymphoproliferative disorder of nk cells to distinguish it from t - cell lgl leukemia as well as aggressive nk - lgl leukemia . the clinical and molecular data presented here suggests that this distinction amongst the chronic lgl diseases may need to be reconsidered . yiyi yan designed and performed experiments , analyzed the data , wrote the manuscript and provided figures . thomas olson designed the tcr qrt - pcr experiments and stat3 mutation detection , analyzed the data , and wrote the manuscript . susan bell nyland designed the tcr deep sequencing experiments , analyzed the data , wrote the manuscript and provided figures .
large granular lymphocyte ( lgl ) leukemia is a chronic clonal lymphoproliferative disorder . here , a t - lgl leukemia patient developed nk - lgl leukemia with residual leukemic t - lgl . 
 tcrv usage and cdr3 sequence drifts were observed with disease progression . 
 a stat3 s614r mutation was identified in nk but not t - cells in the mixed leukemic stage . 
 multiple , non - dominant t - cell clones with distinct stat3 mutations were present throughout . 
 our results suggest that t and nk - lgl leukemia may share common pathogenesis mechanisms and that stat3 mutation alone is insufficient to bring about clonal expansion . 
 mutational and immunological monitoring may provide diagnostic and therapeutic significance in lgl leukemia .