Rituximab is an anti-CD20 chimaeric murine/human monoclonal antibody with significant activity in both de novo and relapsed low-grade and aggressive B-cell non-Hodgkin's lymphoma (NHL) (Maloney et al, 1997; Coiffier et al, 1998, 2002; Colombat et al, 2001). Recently, re-treatment with rituximab for NHL relapsing after previous rituximab therapy has also been shown to be clinically efficacious, with response rates of approximately 40% (Davis et al, 2000). Although the mechanism of resistance to rituximab re-treatment in non-responding patients is unknown, it is possible that selection of CD20-negative NHL could be important in some patients. In support of this theory, a handful of cases detailing the occurrence of CD20-negative NHL relapsing after prior rituximab have been described (Kinoshita et al, 1998; Maloney et al, 1998; Davis et al, 1999; Schmitz et al, 1999; Venugopal et al, 1999). However, the incidence of this phenomenon remains poorly defined. To further define the incidence and nature of CD20-negative relapses occurring after rituximab therapy in aggressive NHL, we retrospectively reviewed the outcome of all patients with CD20-positive aggressive B-cell NHL treated with rituximab at our institution.
Summary. Re-treatment with rituximab for B-cell non-Hodgkin's lymphoma (NHL) relapsing after previous rituximab therapy has recently been shown to be clinically efficacious. Although the mechanism of resistance to rituximab re-treatment in non-responding patients is unknown, it is possible that loss of CD20 expression in the relapsed NHL could be important in some patients. We examined the incidence and nature of CD20 negative relapses following rituximab therapy in aggressive B-cell NHL treated at our institution. Of a total of 18 patients who received rituximab, 13 have relapsed, with 10 patients subsequently undergoing repeat tissue biopsy. Six of these 10 patients (60%) were shown to have lost CD20 expression by either immunohistochemistry and/or flow cytometry. Furthermore, three of the six patients who relapsed with CD20-negative NHL also suffered relapses at unusual anatomical sites. We conclude that loss of CD20 expression in aggressive B-cell NHL relapsing post-rituximab therapy is common. As such, repeat tissue biopsy should be undertaken to document CD20 expression by both flow cytometry and immunohistochemistry prior to considering repeated courses of rituximab in relapsed aggressive lymphomas.
Patients and methods
All patients with biopsy-proven CD20-positive aggressive B-cell NHL (World Health Organization classification, diffuse large B-cell lymphoma and mantle cell lymphoma) (Harris et al, 1999) who had received rituximab therapy at our institution prior to June 2001 were retrospectively reviewed. Indications for rituximab were concomitant low-grade lymphoma, chemorefractory disease, the presence of serious intercurrent illness precluding cytotoxic chemotherapy, and the compassionate use in multiply relapsed, heavily pretreated patients.
Biopsy was taken at disease progression for confirmation of relapse whenever possible. CD20 expression was determined by both flow cytometry and immunohistochemistry if possible. Flow cytometry was performed by standard three-colour direct immunfluorescence using monoclonal anti-CD20 (Immunotech CD20PE, clone B9E9), with list mode data collected on a Coulter Elite ESP Flow Cytometer (Coulter Electronics, Miami, FL, USA). Isotype-negative controls were run with each sample to help set negative/positive voltage thresholds. CD20 expression was considered positive if at least 20% of the cells of interest were labelled with anti-CD20.
Paraffin-embedded tissue sections were used for immunohistochemistry studies (Dako anti-CD20, clone L26). These were performed via high temperature antigen retrieval and a streptavidin-biotin-peroxidase method, as previously described (Kennedy et al, 2002). Positive (sectioned tonsil or appendix) and negative controls were performed with each batch of immunohistochemistry studies. Staining was considered positive if the great majority of malignant cells displayed membrane staining with anti-CD20. Negative staining was reported if complete lack of CD20 expression occurred. In samples in which only a minority of malignant cells expressed CD20, the percentage of malignant cells labelling with anti-CD20 was reported.
A total of 18 patients with aggressive CD20-positive B-cell NHL treated with rituximab were identified. Thirteen patients relapsed or progressed following rituximab therapy; 10 patients with relapsed disease had a repeat biopsy performed. Six of the 10 patients (60%) who underwent repeat tissue biopsy at relapse demonstrated loss of CD20 expression by either flow cytometry and/or immunohistochemisty compared with diagnostic/pre-rituximab biopsies.
Clinical details of the six patients who relapsed with CD20-negative NHL are given in Table I. Median age was 62 years (range, 38–71 years). All patients received rituximab at doses of 375 mg/m2; three patients (cases 1, 2 and 6) received × 4 weekly doses; one patient (case 4) a single dose; one patient (case 3) × 7 doses at approximately 4-weekly intervals; and one patient (case 5) × 10 doses over a 14-month period. Median time to relapse after the last dose of rituximab was 5 months (range 1–12 months). Three patients (cases 2, 3 and 4) had relapses at unusual anatomical sites including a subpectoral mass, quadriceps mass, subcutaneous nodules and trochlear node disease.
|Response to |
|Time to CD20 |
|1||66||Female||DLBCL with discordant |
bone marrow histology
|2nd complete |
|2||71||Female||DLBCL with discordant |
bone marrow histology
|2nd complete remission||Not applicable||6|
|3||58||Male||DLBCL transformed |
|Partial remission |
|4||38||Female||AIDS-related DLBCL||4||Modified CHOPx2||Progressive |
|5||49||Female||Mantle cell lymphoma||1||CHOPx7 |
|2nd relapse||Minimal |
|6||67||Male||DLBCL transformed |
|Stable disease |
All six cases with relapsed CD20-negative NHL were CD20 positive on initial biopsies taken prior to rituximab therapy (Table II). At relapse and/or progression post-rituximab therapy, four patients (cases 1, 3, 5 and 6) demonstrated lack of CD20 expression on both flow cytometry and immunohistochemistry; one patient (case 4) had loss of CD20 demonstrable by immunohistochemistry only (with flow cytometry uninterpretable due to technical reasons); and one patient (case 2) had lack of CD20 expression demonstrable by flow cytometry only (with persistent CD20 positivity on immunohistochemistry).
|Case||Biopsy pre-rituximab||Biopsy post-rituximab|
|1||Cervical node||DLBCL||CD20+||CD20+||Axillary node||DLBCL||CD20–||CD20–|
|2||Axillary node||DLBCL||CD20+||CD20+||Trochlear node†||DLBCL||CD20+||CD20–|
|3||Chest wall||DLBCL||CD20+||CD20+||Subpectoral mass |
|Not done |
|4||Jugulo-digastric node||DLBCL||CD20+||CD20+||Quadriceps mass||DLBCL||CD20–||Technically inadequate sample|
|5||Peripheral blood||Circulating |
|Not available||CD20+||Post-nasal space||Mantle cell |
|Majority (85–90%) |
|Right tonsil||Mantle cell |
|Not available||CD20+||Bone marrow||Lymphoma||Not available||CD20–|
|DLBCL||CD20+||CD20+||Peripheral blood||Circulating |
on cell pellet
Our data suggests that loss of CD20 expression at relapse following prior rituximab therapy for aggressive B-cell NHL is common, occurring in 60% of patients who underwent repeat tissue biopsy in our series. Given the potential benefit of re-treatment with rituximab in relapsed B-cell NHL, our observation has significant clinical importance, suggesting that repeat tissue biopsy should be performed to document the presence (or absence) of CD20 expression prior to considering repeated courses of rituximab for relapsed aggressive NHL.
Two different patterns of CD20 negativity at relapse were observed in our series. Firstly, in five patients (cases 1, 3, 4, 5 and 6), loss of CD20 expression was observed on immunohistochemistry +/– flow cytometry, as has been previously described in the published cases documenting this phenomenon (Kinoshita et al, 1998; Maloney et al, 1998; Davis et al, 1999; Schmitz et al, 1999; Venugopal et al, 1999). Secondly, in one patient (case 2), lack of CD20 expression was demonstrable only by flow cytometry, with persistent CD20 expression present on immunohistochemistry stains. This second pattern of CD20 negativity has been suggested to simply represent bound rituximab blocking surface CD20 binding sites, resulting in only apparent negativity by flow cytometry (Grillo-Lopez & Kunkel, 2000). However, even if bound rituximab was responsible for the loss of demonstrable CD20 expression on flow cytometry, the continued survival and proliferation of the lymphoma in this case suggests that it was nevertheless resistant to its cytotoxic effects. As such, we believe that recognition of this pattern of CD20 negativity at relapse is still clinically important if re-treatment with rituximab is under consideration. An alternative hypothesis is that this second pattern of CD20 negativity represents selection of lymphoma clones with loss of and/or mutated surface CD20 domains, as demonstrated by the different epitopes recognized by the anti-CD20 monoclonal antibodies used in flow cytometry (extra-cellular/surface epitopes) and immunohistochemistry (intracytoplasmic epitopes). We were unfortunately unable to prove or disprove this possibility.
The incidence of relapsed CD20-negative NHL seen in our series was markedly greater than reported in the literature. However, in most series commenting on this phenomenon, either the specific histology of the treated lymphomas was not detailed (Davis et al, 1999; Grillo-Lopez & Kunkel, 2000) or a variety of B-cell lymphomas, including low grade NHL, were included (Foran et al, 2001). Our review was restricted to relapses occurring after rituximab therapy in aggressive B-cell NHL. It is also noteworthy that the published case reports of CD20-negative NHL occurring after prior rituximab therapy have all involved transformed histologies; to our knowledge no cases of relapsed CD20-negative indolent NHL have been reported (Kinoshita et al, 1998; Davis et al, 1999; Schmitz et al, 1999; Venugopal et al, 1999). As such, it is probable that the high incidence of CD20-negative relapsed NHL seen in our series relates to the aggressive nature of the lymphomas being treated. However, further study is still required to more accurately define the risk of this phenomenon at relapse in both low-grade and aggressive NHL.
The selection and/or evolution of clonal tumour cells with mechanisms of resistance to specific antineoplastic agents upon exposure to those agents are well recognized (Meeker et al, 1985). Previous studies have documented that measurable circulating rituximab can persist for up to 6 months after treatment (Berinstein et al, 1998). This potential for prolonged tumour exposure to circulating rituximab could potentially increase selection pressure for the emergence of resistant clones. To this end, it is noteworthy that the CD20-negative relapse in case 4 of our series occurred after only a single rituximab dose. We were unfortunately unable to determine whether rituximab exposure selected a pre-existing CD20-negative clone for subsequent outgrowth or, in fact, induced evolution of a clone to lose CD20 expression. The number of CD20-negative relapses observed at unusual anatomical sites in our series does suggest that evolution of new clones may play a role.
In conclusion, we have shown a high incidence of CD20-negative relapses occurring following use of rituximab in aggressive B-cell NHL. Despite the preliminary nature of our findings, given the potential benefit of re-treatment with rituximab in relapsed B-cell NHL, we recommend that repeat tissue biopsy be undertaken to document CD20 expression by both flow cytometry and immunohistochemistry prior to considering repeated courses of rituximab in relapsed aggressive lymphomas.