Homozygous FCGR3A‐158V alleles predispose to late onset neutropenia after CHOP‐R for diffuse large B‐cell lymphoma

Recent reports suggest genetic polymorphisms influence susceptibility to rituximab‐induced late‐onset neutropenia (LON), which in turn may be a predictor of good outcome in B‐cell lymphoma.


Introduction
Diffuse Large B-cell Lymphoma (DLBCL) is the commonest aggressive B-cell lymphoma. Rituximab is a routine component of DLBCL therapy, typically as part of 'CHOP-R' (cyclophosphamide / hydroxydaunorubicin / Oncovin [vincristine] / prednisone / rituximab). It has resulted in a marked improvement in response and survival. 1 The importance of host genetics on the mode of action of rituximab in DLBCL is unclear.
Two principle mechanisms of action are postulated. The first is antibody dependent cellular cytotoxicity (ADCC) whereby rituximab binds to FCGamma Receptor (FCGR) bearing Natural Killer (NK) cells, resulting in destruction of CD20 + normal and malignant B-cells by the reticulo-endothelial system. [2][3][4][5] The other is direct lysis via complement dependent cytotoxicity (CDC). [6][7][8][9][10] FCGR3A is a low-affinity receptor capable of binding the FC portion of complexed but not monomeric IgG. A polymorphism at amino acid 158, alternatively encoding for a valine (V) or phenylalanine (F), has been identified. FCGR3A-V158 has a higher binding affinity for IgG 1 than FCGR3A-F158. 11 There is evidence that this polymorphism is important in the treatment of colon and breast cancers with cetuximab and transtuzumab respectively. 12,13 Data on the impact of the FCGR3A-V158F polymorphism in DLBCL treated with CHOP-R is conflicting. 14,15 There are also reports that the polymorphism may contribute to the development of late-onset neutropenia (LON). 16,17 This is a relatively rare but well-recognized late complication of rituximab containing therapy, defined as grade 3-4 neutropenia occurring after neutrophil recovery from last therapy in the absence of other causes. Although not designed for survival analysis, it is notable that in previous case series lymphoma patients with LON have a strikingly low incidence of disease progression. It has therefore been proposed that development of LON is a measure of good outcome, perhaps reflecting enhanced potency of rituximab. 18 Binding of C1q to the Fc portion of immune complexes activates CDC through initiation of the complement cascade. C1q is encoded by C1qA, whose sole coding polymorphism is at position 276, coding for adenine (C1qA-A276) or guanine (C1qA-G276). C1qA-A276 results in lower C1q protein levels than the C1qA-G276 polymorphism. Breast cancer patients heterozygous or homozygous for the C1qA-G276 genotype have a higher rate of metastasis. 19 In a study of 133 patients with follicular lymphoma treated with single agent rituximab, possession of the C1qA-A276 allele was associated with increased response rates and prolonged response duration, even after adjusting for FCGR3A-V158F polymorphisms. 20 The role of C1qA polymorphisms in DLBCL has yet to be evaluated.
Our study is to our knowledge the first examining the influence of C1qA-A276G polymorphism in DLBCL. It is also the largest to date to assess FCGR3A-V158F polymorphisms in DLBCL treated with CHOP-R, and the first to assess the influence of either polymorphism on both outcome to chemo-immunotherapy and susceptibility to LON.

Methods
Analysis was restricted to DLBCL patients treated with CHOP-R between 1 st Inclusion criteria for LON was neutrophils <1000/μl occurring greater than six weeks after receiving last rituximab, with no other identifiable causes of neutropenia.
Patients with human immunodeficiency virus infection, pelvic irradiation or marrow involvement at restaging were excluded. Follow-up evaluation (included full blood counts) was performed at least every three months for the first two years.
DNA was extracted from FFPE tissue (patients) or buccal scrapes (controls) using standard procedures and analysis performed in batches. All samples were analyzed in the same laboratory. FCGR3A-V/F158 and C1qA-G/A276 genotyping were performed using allele specific PCR based on protocols previously described. 11,22 . In selected samples results were confirmed by directed sequencing.
Event-free and overall survival (EFS and OS) were estimated using the Kaplan-Meier product-limit method. EFS was measured from the date of diagnosis to first documented progression, change to alternate therapy, death, or the last follow-up visit.
OS was calculated from the date of diagnosis to death from any cause or the last follow up. Survival rates were compared for statistical differences by using log-rank analysis. P values less than 0.05 were considered statistically significant and all correspond to 2sided significance tests. The chi-squared and Fishers exact tests were used to compare clinical and laboratory parameters between the different polymorphisms. All statistics were performed on the Graphpad Prism platform (version 5). year OS rates were 71% and 77%, respectively. The response to treatment with CHOP-R is comparable to published results. 23 As expected, the international prognostic index  In addition, because LON is not diagnosed until six weeks after chemo-immunotherapy, this may indicate that these patients do not have refractory disease which would skew towards their having a more favourable prognosis.

Patient characteristics by FCGR3A-V158F and
We observed a 6% incidence of LON in DLBCL patients treated with CHOP-R chemo-immunotherapy. Previous reports of LON after induction chemoimmunotherapy for B-cell lymphoma have not been restricted to a single histological sub-type or treatment regimen. In addition the neutrophil cut-off used to identify LON has varied between grade 2 to grade 4. [24][25][26] Once studies are restricted to those using a definition of grade 3/4 neutropenia, then irrespective of ethnicity the incidence of LON (range 7-13%) is of a similar order of magnitude to our series. 16,27,28 In these as with our study, incidence may be underestimated as patients are frequently asymptomatic.
The incidence of LON appears to rise markedly in studies that include patients undergoing high-dose therapy with autologous stem cell rescue. 17