Historically, LGLL could be readily recognized by reviewing a PB smear. An LGL count of more than 2 × 109/L (normal LGL count in PB: 0.2–0.4 × 109/L) lasting > 6 months, was considered a criterion for determining this disease.[12, 22] For T-LGLL, the current diagnostic requirements have lowered this threshold to > 0.4 or 0.5 × 109/L provided that a clonal T-LGL population is found with an appropriate clinical context.[23–25] Recent studies have shown that 49% of patients with T-LGLL have no absolute lymphocytosis and 36% of patients have blood LGLs < 1 × 109/L. As the clinical manifestations of RA-associated T-LGLL are often identical to those in which one would suspect an FS, it may be difficult to differentiate RA-associated T-LGLL with a low LGL count (0.4–2.0 × 109/L) from FS. Moreover, expansion of LGLs can be detected in patients with FS.[26–28] RA-associated T-LGLL and FS can be distinguished by T-cell clonality determined by assessing the TCR gene rearrangements present in T-LGLL but not in FS.[1, 5, 29] However, there is considerable discussion regarding the significance of dominant T-cell clones as a hallmark of T-cell malignancy because small populations of clonally expanded T-LGLs are revealed in healthy individuals and in an exuberant reactive response.[30–34] Considering that the difference between RA-associated T-LGLL and FS often depends on a single test with well-known gray areas in interpretation and limitations, it is necessary look for additional distinctions. We can use mutations in STAT3 and STAT5b genes as molecular markers for T-LGLL diagnostics, but their prevalence in FS and their diagnostic value for differential diagnosis between FS and RA-associated T-LGLL are unclear. In this study, we did not detect STAT3 mutations in any of the 24 cases with FS, as opposed to 22 of 56 patients with RA-associated T-LGLL. Further, no STAT5b mutation was detected in any FS or RA-associated T-LGLL patient in our cohort.
Savola et al. examined STAT3 and STAT5b mutations in 14 patients with RA and neutropenia. Similar to our patient cohort, they did not find any STAT5b mutations. However, in contrast to our results, they identified STAT3 mutations in 6 of 14 (43%) patients. We believe that difference between outcomes obtained by Savola et al. and our study can be attributed to different methods of assessing T-cell clonality and the patient selection criteria. We tested T-cell clonality based on the rearrangement of gamma and beta chain-encoding genes by a PCR-based assay, whereas Savola et al. studied the clonality of T cells by flow cytometry using a Vβ kit, covering only 70% of the Vβ T-cell repertoire. In contrast to Savola et al., we did not include patients with T-cellular clonality in the FS group.
Female prevalence, age at FS diagnosis, and duration of RA prior to FS diagnosis in our series were comparable to the results found in literature.[4, 5, 38] Overall, in our patient cohort, RA was of moderate activity, even though RA is typically severe in patients with FS. All our patients were seropositive: RF+/anti-CCP+/anti-MCV + or RF−/anti-CCP+/anti-MCV+. Splenomegaly ranging from massive to detectable only based on abdominal imaging modalities, was detected in 83% of patients. SS was diagnosed in our study in 28% of patients, which is significantly less than in the FS patient cohorts reported by other authors: 48% (Sienknecht et al.), 69% (Barnes et al.), and 53% (Campion et al.).[2, 3, 39]
In our study, low count expansion of LGLs (0.4–2.0 × 109/L) in PB was detected in 56% of cases, but did not exceed 2.0 × 109/L and the bone marrow aspirate differential count showed no increase in lymphocytes. Flow cytometric immunophenotyping studies play an important role in the diagnosis of T-LGLLs. The expression of CD57 and CD16 antigens, one or both of which are detected in the vast majority of T-LGLL cases, was found on cytotoxic T-lymphocytes in only 56% and 7% of our cases, respectively. In contrast, aberrant expression of CD5 was the most common finding in our patient group. This abnormality is frequently associated with T-LGLL, but is also found in T-cell reactive expansion.[41, 42] Bone marrow involvement is present in at least 75% of T-LGLL cases, although it is often subtle and difficult to detect. Specific criteria have been proposed for the diagnosis of T-LGLL in bone marrow sections using immunohistochemistry.[42; 43] However, as reported by Burks et al., there are probably no distinctive features in bone marrow biopsies that would separate T-LGLL from FS. In 2 patients with FS in our cohort, immunohistochemical studies also revealed that bone marrow infiltration by cytotoxic T-lymphocytes was indistinguishable from T-LGLL lesions.
The pathogenesis of neutropenia in FS has not yet been fully studied and seems to be multifactorial. In 12 of 14 cases in our study, the bone marrow aspirate differential count fit into the expected consequence of peripheral destruction/sequestration of neutrophils. Although the role of splenic sequestration/destruction in neutropenia pathogenesis is not supported by all studies, splenectomy produces a long-term hematologic response in 80% of patients with FS. We also observed persistent recovery of neutrophil levels after splenectomy in 2 of our patients with FS and massive splenomegaly.
We are aware of some limitations concerning our study design. Due to the retrospective design of the study, some of the data were incomplete. Additionally, we used of allele-specific TaqMan real-time PCR rather than Sanger sequencing to detect somatic point mutations in STAT3 and STAT5b genes. A set of primers for most common mutations in STAT3 and STAT5b genes was developed. Even though this approach provides much higher sensitivity compared to Sanger sequencing, some rare mutations, not covered by the developed primers, could not be identified.