Diffuse large B-cell lymphoma represents approximately 30%–40% of all diagnoses of non-Hodgkin’s Lymphoma and may represent up to 80% of all lymphomas that arise in the palatine tonsils. Several studies have attempted to correlate clinical, laboratorial, and tissue factors with the prognosis of the lymphomas, such as the International Prognostic Index, the tissue expression of some proteins, and the lymphocyte count at the time of diagnosis, as well as to correlate Epstein-Barr virus (EBV) infection with worse prognoses. Patients with palatine tonsil DLBCL, from Salvador, Bahia, Brazil, were studied in order to identify prognostic factors. Twenty-four patients with DLBCL were studied. The factors that negatively influenced the patients’ survival rates were the lymphocyte count at the time of diagnosis <1.000/mm3 and the Bcl-2 protein expression. There was no CD5 expression in these lymphomas, and neither was there an association with EBV infection.
The palatine tonsils, along with the nasopharyngeal lymphoid tissue, the base of the tongue, and the oropharyngeal wall make up Waldeyer’s ring. This ring is located at the entrance of the respiratory and digestive tract, being the second most common site of extranodal lymphomas, after the gastrointestinal tract [
Most lymphomas found in the palatine tonsils are the B-cell type, and, of these, diffuse large B-cell lymphoma (DLBCL) represents most of the cases, reaching as much as 80% in some of the groups studied [
Although morphologically indistinct, some molecular studies support the hypothesis that DLBCL makes up a heterogeneous group of lymphomas that has different prognostic implications [
Another controversial prognostic tissue marker has been the detection of Epstein-Barr virus (EBV) infection in neoplastic cells. While in pediatric Hodgkin’s Lymphoma this infection is associated with a better prognosis [
Considering that the use of IPI has been shown to be insufficient as the only prognostic marker in DLBCL, we evaluated other laboratory and tissue markers in patients with DLBCL of palatine tonsils, who came from a reference institution for the diagnosis and treatment of patients with cancer in Northeastern Brazil.
The patients were selected at the Pathological Anatomy Service of the Aristides Maltez Hospital in Salvador, Bahia, Brazil. All patients with a diagnosis of DLBCL of the palatine tonsils were included. The diagnoses were carried out between January of 1999 and December of 2006, on patients with illness primary site in the tonsils, or on those who presented the main tumor mass in the same area.
The diagnoses were reviewed by a hematopathologist (I.A.) according to the criteria established by the lymphoid neoplasia classification of the World Health Organization 2008 [
The tissue used in the study had been set in formalin. Serial 4
Antibodies used for immunohistochemical study.
Antibody | Clone | Dilution | Source |
---|---|---|---|
CD3 | F7.2.38 | 1 : 100 | DakoCytomation |
CD5 | 4C7 | 1 : 50 | Novocastra |
CD10 | 56C6 | 1 : 50 | Novocastra |
CD20 | L26 | 1 : 100 | DakoCytomation |
Bcl-2 | 124 | 1 : 50 | DakoCytomation |
Bcl-6 | P1F6 | 1 : 20 | Novocastra |
MUM1 | MUM1p | 1 : 50 | DakoCytomation |
p53 | PAb1801 | 1 : 100 | Novocastra |
Ki67 | MIB-1 | 1 : 50 | DakoCytomation |
For the antibodies CD3, CD5, CD10, CD20, Bcl-2, Bcl-6, and MUM1, the cases with more than 10% of the tumor cells marked were considered positive. For the Ki-67 analysis, the cases were semiquantified on a positivity scale of 0 to 100%, according to the quantity of marked tumor cells per field, in an increase of 400x. The p53 protein analysis was carried out using the Sannino and Shousha score [
The IHC classification according to the Hans et al. algorithm [
In situ hybridization for the detection of EBER 1 and 2 transcription of EBV was carried out in RNA-free conditions, using specific probes marked with digoxigenin, as previously described [
To compare the difference between the two proportions, the chi-square test and Fisher exact test were used. The differences between two means were analyzed by the Mann-Whitney test. The Kaplan-Meier survival analysis and the log-rank test were used to study the prognostic significance of the utilized biomarkers. The overall survival (OS) was calculated from the date of diagnosis to the last evaluation, or date of death. Event-free survival (EFS) was calculated from the date of diagnosis until date of death, disease progression, or end of clinical followup. The
During the time of the study 567 diagnoses of non-Hodgkin’s Lymphoma were made in the study institution. Of these, 253 were classified as DLBCL (44.6%). Twenty-six diagnoses of DLBCL of the palatine tonsils were made (4.6% of the total amount of DLBCL), and for this present work 24 cases of DLBCL of the palatine tonsils were studied, due to the exclusion criteria. Table
Clinical features of the patients.
Parameters | Frequency (%) |
---|---|
Gender | |
Male | 11 (45.8) |
Female | 13 (54.2) |
Age (mean) | 60 (range 15–86) |
Stage | |
I/II | 13 (45.8) |
III/IV | 6 (25) |
B symptoms | 12 (50) |
Performance status | |
<2 | 23 (95.8) |
≥2 | 1 (4.2) |
Serum LDH | |
Normal | 16 (66.7) |
Elevated | 2 (8.3) |
IPI | |
0 and 1 | 15 (62.5) |
2 | 2 (8.3) |
3 | 1 (4.2) |
4 and 5 | 0 |
LDH: lactate dehydrogenase; IPI: International Prognostic Index.
Most of the patients were treated with a schema based on anthracycline (CHOP or similar ones). The average follow-up time of the patients was 43 months (range 1 to 104 months). The overall and event-free survival rates were on average of 43.5 months to 39.5 months, respectively. Most of the patients presented a low-risk IPI (Table
The mean lymphocyte count at the time of diagnosis was 1.980 cells/mm³, varying between 354 and 3.922 cells/mm³, and 18.2% of the patients presented a lymphocyte count <1.000 cells/mm³. Patients with a lymphocyte count at the time of diagnosis ≥1.000 cells/mm³ presented OS (74.9 months versus 16 months) and EFS (74.7 months versus 8.2 months) significantly greater than patients with a count lower than this value (
Overall survival and event-free survival considering lymphocyte count at the time of diagnosis.
According to the algorithm of Hans et al. [
Immunohistochemical aspects of the palatine tonsils DLBCL: positivity for CD10 (a), Bcl-6 (b), MUM-1 (c), and Bcl-2 (d) antibodies.
The Bcl-2 protein expression (Figure
Overall survival and event-free survival considering Bcl-2 protein status at the time of diagnosis.
The p53 protein expression was found in 45.8% of the patients with predominance of the male gender (
All the patients presented negative in situ hybridization for EBER-1 and 2 transcription of EBV.
Among the non-Hodgkin’s lymphomas, we observed a DLBCL frequency similar to other studied series; however, the palatine tonsils were mostly attacked at a slightly greater frequency than that referred to in the literature [
We observed a predominance of tonsil lymphoma patients and low-risk IPI. When observed alone, the IPI did not show any significance to predict survival rates, and this index might not be the most adequate prognostic factor for patients with extranodal lymphoma. However, the reduced number of patients in this study may have had an influence on this analysis.
Recent studies have shown the lymphocyte count at the time of diagnosis with a prognostic factor in DLBCL [
DLBCL has several forms of presentation and is a heterogeneous entity seen as certain IHC marker expressions, which could give it a better or worse prognosis. Hans et al. [
In a more consistent manner, the Bcl-2 expression in the pre-Rituximab era has proven to be an unfavorable prognostic factor [
Still quite a controversial issue is the prognostic value of the p53 protein expression in patients with DLBCL. In two other Brazilian studies that researched this correlation [
In the present study all the patients were negative for the CD5 marker. The expression of this marker has been reported as between 5%–10% of the patients who have
Several studies in the literature have demonstrated the association between EBV infection and some subtypes of non-Hodgkin’s lymphoma [
No association was found between EBV infection and DLBCL in the patients of this study. This data is different from what was found by Park et al., who studied 380 patients with DLBCL, where 9% presented EBV infection [
We conclude that, in our midst, the palatine tonsil diffuse large B-cell lymphomas predominantly presented a non-GC profile and were not associated with EBV infection. The factors that negatively influenced the OS and EFS rates of these patients were the lymphocyte count at the time of diagnosis <1.000/mm³ and the Bcl-2 protein expression. New studies are necessary to attempt to explain the etiology of these lymphomas and to identify other prognostic factors.
There is no conflict of interests for any of the authors involved in this study.
The authors wish to thank all the employees of the Pathological Anatomy Service of the Aristides Maltez Hospital and Dr. William Harrington, Jr. (in memoriam) for their help in obtaining the immunohistochemical markers.