Germline Mutations in CTLA4 and Spectrum of Bone Marrow Features Overlapping with Aplastic Anemia and Large Granular Lymphocyte Lymphocytosis

Introduction: CTLA4 haploinsufficiency due to germline heterozygous mutations in the cytotoxic T-lymphocyte antigen-4 gene (CTLA4) results in severe systemic immune dysregulation; infiltrative lymphocytic lesions of lungs (e.g. lymphocytic pneumonitis), GI tract (e.g.lymphocytic colitis), and CNS; and a spectrum of autoimmunity, immunodeficiency and peripheral blood cytopenias. We describe the hematologic, bone marrow (BM), and flow cytometry (FC) features of CTLA4 haploinsufficiency of a cohort of 14 patients, which show overlapping morphologic features with acquired aplastic anemia (acAA), and large granular lymphocyte lymphocytosis (LGL) in a subset of patients. We compared the findings to those in a separate cohort of 20 age matched patients diagnosed with acAA. There were significant differences between acAA and CTLA4 deficiency based on analysis of BM lymphocyte subsets and marrow lymphocyte infiltration patterns.

Materials and Methods: Peripheral blood (PB), BM and FC analysis of patients with germline mutations in CTLA4, and acAA was performed along with review of clinical, molecular and laboratory data. Statistical comparisons were made using Mann Whitney test, with p < 0.05 for significance.

Results: Median age at initial evaluation for patients with CTLA4 haploinsufficiency was 23.5 years (range 11-71 years), with a male-to-female ratio of 11:3. 11 out of 14 patients had cytopenias. Of those with cytopenias 2/11 had pancytopenia, 4/11 had bicytopenia and 5/11 had unilineage cytopenia. Hypogammaglobulinemia was common (13/14). In some cases cytopenias were recognized as autoimmune in etiology with immune thrombocytopenic purpura and autoimmune hemolytic anemia, among the most common hematologic manifestations. Three patients met criteria for a diagnosis of moderate AA, 1 for severe AA, and 1 for LGL. Overall bone marrow biopsies were hypocellular for age (10/14) with a subset (29% [4/14]) showing severe trilineage hypoplasia with ≤20% cellularity. Atypical interstitial CD8-positive T-cell infiltrates with prominent non-paratrabecular CD4-positive T-cell aggregates were evident by immunohistochemistry in most cases (10/14), accompanied by a near absence of B-cells. T-cell clonality was assessed by T-cell receptor (TCR) gamma PCR and showed abnormal oligoclonal T-cell populations in 3/8, and one case with a clonal T-cell receptor gene rearrangement. B-cell IgH PCR reactions were polyclonal. In all cases, cytogenetic studies were either negative or showed only minor abnormalities (i.e. loss of Y chromosome). Given the morphologic overlap of a subset of CTLA4 cases with acAA, we compared bone marrow flow cytometry analysis of 8 CTLA4 cases with 20 acAA cases. CTLA4 patients showed >5 fold reduction in the median percentage of B-cells in comparison to acAA (p=0.0005). While acAA cases demonstrated relative preservation the B-cell compartment with intact B-cell maturation sequence; in contrast CTLA4 cases showed abnormal B-cell maturation patterns in half of cases characterized by few detectable early B-cell precursors, and progressive loss of intermediate and mature forms. T-cell populations in CTLA4 showed inverted CD4:CD8 ratios with relatively increased CD57+ T-cells, and atypical dim expression of CD20 on T-cells. Morphologically, when the markedly hypocellular CTLA4 cases were compared to acAA on core biopsies, little difference in marrow cellularity was seen; however, the CTLA4 cases showed multiple large atypical T-cell aggregates and near total absence of B-cells, which was not seen in acAA; acAA cases showed scattered background T-cells and B-cells.

Conclusions: CTLA-4 deficiency presents with cytopenias, autoimmunity, and immunodeficiency with a subset of patients that have features overlapping with bone marrow failure, specifically AA and LGL. Herein we demonstrate a combination of abnormal BM findings, previously unreported, comprising hypocellularity, CD8-positive interstitial T-cell infiltrates with atypical CD4-positive T-cell aggregates, marked B-cell lymphopenia, increased LGLs and abnormal T-cell clonality patterns by PCR. These features may help identify patients harboring CTLA4 mutations for early detection and optimal therapeutic management.