Late-Onset Neutropenia in Rituximab-Treated Lymphoma Patients: Lymphocyte Subpopulation Imbalances, Bone Marrow Hematopoiesis and Immunohistology.

Late-onset neutropenia (LON) is a complication of Rituximab of yet unknown pathophysiology. We investigated potential underlying mechanisms in 12 patients with various non-Hodgkin lymphoma (NHL) subtypes who developed LON without identifiable causes at a median of 95 (range 67–420) days after completion of the intended treatment with Rituximab ± chemotherapy. The study also included two control groups:

1. healthy donors (n=25) for comparative analysis of bone marrow (BM) functional parameters;

2. NHL patients treated with similar Rituximab ± chemotherapy regimens without LON for comparative evaluation of peripheral blood and BM lymphocyte subpopulations (n=38) and BM pathology findings (n=27).

Ten of 12 patients with LON and 33/38 NHL controls developed profound B-cell depletion. Inverted CD4/CD8 cell ratios were observed in 10/12 LON cases vs. 13/38 NHL controls (p<0.01). A rise in CD8 cell count >1.0x10⁹/L was identified in 8/12 LON cases vs. 8/38 NHL controls (p<0.01). T-large granular lymphocyte proliferation (CD3+CD8+CD57+ cells >30%) was observed in 7/12 LON cases vs. 14/38 NHL controls (p=0.1885). BM biopsy samples from 10/12 LON cases were examined at onset of neutropenia. Mild to moderate lymphocytic infiltration with predominantly nodular and/or interstitial growth was observed in all cases. Lymphoid aggregates lacked CD20+ or CD79a+ B-cells and were composed entirely of CD3+CD45RO+CD43+ T-cells. In the control group, BM infiltration by T cells with similar features as above was observed in 18/27 cases. Seven of 10 LON patients showed moderate-to-significant hypoplasia of the granulocytic series; the remaining three exhibited hyperplasia. All ten LON patients had pronounced shift to the left, extending to maturation arrest in 6/10 cases. In contrast, 21/27 NHL control cases (78%) showed granulocytic hyperplasia; the remaining cases were either normal or showed granulocytic hypoplasia. As in the LON cases, the control NHL cases exhibited different degrees of shift to the left of the granulocytic series. Hyperplasia of the erythroid and megakaryocytic series with dyserythropoiesis and dysmegakaryopoeisis, respectively, was identified in all LON cases and most NHL controls. In all cases with repeat samples, MDS-like changes eventually resolved. Compared to healthy donors, LON patients displayed:

1. low reserves of granulocytic progenitors associated with increased apoptosis and increased proportion of Fas-expressing cells within the CD34+/CD33+ cell compartment;

2. Fas-Ligand and Interferon-γ mRNA expression within BM CD3+ cells;

3. increased TNFα and IL-1β levels in long-term BM culture supernatants;

4. accelerated apoptosis in all stages of BM granulocytic development.

All LON patients were negative for granulocyte-reactive antibodies. We conclude that T-cell mediated autoimmune myelopathy/myelodysplasia associated with MDS-like changes of all myeloid series and suppression of predominantly granulocytic progenitor cell growth is critically implicated in the pathophysiology of Rituximab-related LON, at least in a subset of cases.