Viral response to chemotherapy in endemic burkitt lymphoma

Abstract

Purpose: Some EBV-directed therapies are predicted to be effective only when lytic viral replication occurs. We studied whether cyclophosphamide chemotherapy induces EBV to switch from latent to lytic phases of infection in a series of EBV-associated Burkitt lymphomas.

Experimental design: Children with first presentation of an expanding, solid maxillary or mandibular mass consistent with Burkitt lymphoma underwent fine-needle aspiration just prior to the initiation of cyclophosphamide therapy and again 1 to 5 days later. Aspirated cells were examined for latent and lytic EBV infection using in situ hybridization to EBV-encoded RNA (EBER), immunohistochemical analysis of the lytic EBV proteins BZLF1 and BMRF1, reverse transcription PCR targeting BZLF1 transcripts, and EBV viral load measurement by quantitative PCR.

Results: Among 21 lymphomas expressing EBER prior to chemotherapy, 9 of 10 still expressed EBER on day 1 after therapy whereas only 2 of 11 (18%) specimens still expressed EBER at days 3 to 5, implying that chemotherapy was fairly effective at eliminating latently infected cells. Neither of the lytic products, BZLF1 or BMRF1, were significantly upregulated at the posttherapy time points examined. However, EBV genomic copy number increased in 5 of 10 samples 1 day after treatment began, suggesting that viral replication occurs within the first 24 hours.

Conclusion: Cyclophosphamide may induce the lytic phase of EBV infection and is fairly effective in diminishing EBER-expressing tumor cells within 5 days. These findings provide the rationale for a trial testing synergistic tumor cell killing using cyclophosphamide with a drug like ganciclovir targeting lytically infected cells.

Figure 1. Cytologic and histochemical features demonstrate loss of EBER-expressing Burkitt lymphoma cells after cyclophosphamide therapy

(A) In a newly diagnosed patient, hematoxylin and eosin stain reveals features of Burkitt lymphoma including atypical lymphoid cells with fine, blast-like chromatin, and a large cell-laden macrophage in the lower left. (B) Immunohistochemistry reveals CD20 localized to cytoplasm and membrane of atypical cells. (C) EBER is expressed by in situ hybridization in many nuclei, whereas smaller lymphoid cells stain only with the eosin counterstain; (D) In an aspirate from the same patient 4 days after start of chemotherapy, EBER is not expressed while a control hybridization targeting oligo-dT (inset) shows RNA is preserved in most cells.

Figure 2. EBV viral load may increase initially but then falls consistently in post-therapy tumor specimens

EBV viral load, expressed in copies per 100,000 cells, is displayed as mean with standard error bars in each clinical subgroup of Burkitt lymphomas. Both pre-treatment groups have similar distributions of viral burden. Mean viral load is even higher among tumors sampled on day one, but has declined by days 3 to 5 after start of chemotherapy.