Splenic diffuse red-pulp small B-cell lymphoma associated with hepatitis B virus: a report of two cases

ABSTRACT CONTEXT: Splenic diffuse red-pulp small B-cell lymphoma is a rare disease, representing less than 1% of all non-Hodgkin lymphomas (NHL). This entity is characterized by involvement of bone marrow sinusoids and peripheral blood. The majority of cases are at an advanced stage when diagnosed. Its pathogenesis is still poorly understood. CASE REPORTS: We report on two patients with chronic non-replicating hepatitis B virus (HBV) who developed splenic diffuse red-pulp small B-cell lymphoma. Both of them were in stage IV at diagnosis and evolved with aggressive disease. Both of them achieved a complete response through chemotherapy, but one of them died due to infectious complications during bone marrow transplantation. The other decided not to undergo transplantation and continues not to show any evidence of disease today (three years after treatment). Some studies have shown a possible association between B-cell NHL and HBV. Nonetheless, the mechanism through which this oncogenic virus interacts with B-cell NHL is still poorly understood. HBV is lymphotropic and may insert into the host’s genome, thus causing overexpression of oncogenes and downregulation of tumor suppressor genes. Therefore, chronic stimulation by HBV can increase B-cell proliferation, which promotes monoclonal expansion of these cells and results in malignancy. CONCLUSION: HBV may be implicated in the pathogenesis of this lymphoma, although no direct association between these two entities could be proved in the present study. Further investigations are necessary.


INTRODUCTION
Splenic diffuse red-pulp small B-cell lymphoma was recognized as a provisional entity in the 2008 update of the World Health Organization (WHO) classification. 1 This new WHO update recognized two categories of primary splenic lymphomas; splenic marginal-zone lymphoma (SMZL) and the provisional group of splenic B-cell lymphoma/leukemia unclassifiable (SLLU), that includes splenic diffuse red-pulp small B-cell lymphoma and hairy-cell leukemia variant (HCL-v). 1 The median age at which patients are diagnosed with splenic diffuse red-pulp small B-cell lymphoma is 72 years (range: 69-74 years), 2 and the disease is seen predominantly among males, with a male/female ratio of 2.4:1.
Splenic diffuse red-pulp small B-cell lymphoma is a rare disease and represents less than 1% of all non-Hodgkin lymphomas (NHL) and 10% of B-cell lymphomas that are described in postsplenectomy series. 1 This malignancy is characterized by diffuse infiltrate of monomorphic small to medium-sized B-cells with cytoplasmic villi, going into the red pulp of the spleen.The bone marrow sinusoids and peripheral blood are frequently involved.
Patients usually present with an advanced stage of disease when they are diagnosed.Peripheral lymph node involvement and B-symptoms are rarely reported and massive splenomegaly is common. 1 Splenic diffuse red-pulp small B-cell lymphomas have an indolent clinical course and there is no known standard treatment approach.So far, most of these patients have been treated with splenectomy. 3e pathogenesis of this lymphoma is still poorly understood and no risk factors associated with its development have been described.Recent studies have shown an association between B-cell NHL and hepatitis B virus (HBV), but the mechanism through which this oncogenic virus results in chronic lymphoproliferative B-cell disorders is not clearly understood.To the best of our knowledge, there is no case report in the literature correlating HBV with splenic diffuse red-pulp small B-cell lymphoma.
Here, we describe two cases of splenic diffuse red-pulp small B-cell lymphoma associated with chronic HBV infection.

CASE REPORTS Case 1
A 62-year-old woman presented at our clinic complaining of weight loss, fever, night sweats and increasing abdominal size over the preceding two months.In her medical history, she reported having had rheumatoid arthritis, which was previously treated with chloroquine and methotrexate, and an untreated chronic non-replicating HBV infection (AntiHbC total +, AgHbe −, AntiHbe +, AgHbs +, AntiHbsAg -) with quantitative polimerase chain reaction (PCR) for HBV of 104 IU/ml (normal range of values: 20-170,000,000 IU/ml).Other viral serological tests for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) were negative.In her family history, her daughter had had a central nervous system (CNS) neoplasm, a 60-year-old brother had had prostate cancer and another 50-year-old brother had died of acute leukemia.
In her physical examination, she presented as emaciated and pale, with massive splenomegaly and no palpable hepatomegaly or peripheral lymphadenopathy.Computed tomography (CT) scans showed subtle hepatomegaly and greatly increased spleen size, with no lymphadenopathy (Figure 1).
Laboratory tests showed normocytic and normochromic anemia (hemoglobin 8.8 g/dl, platelet count of 124 x 10 9 /l and total leukocyte count of 6.18 x 10 9 /l); 21% of the lymphoid cells had a high nuclear-to-cytoplasmic ratio, with loose chromatin, small clear nucleolus and thin cytoplasmic projections.
Immunophenotyping of peripheral blood cells showed that they were positive for CD19, CD20, FMC7, CD22, and CD23, with Splenectomy was performed as a therapeutic procedure and the histological analysis showed that the spleen weighed 1.818 kg, measured 27 x 14.3 x 8.3 cm and presented diffuse infiltrate in the red pulp of the spleen, with both cord and sinusoid infiltration by small B-cell lymphocytes (Figure 2) expressing CD20 and DBA-44 antigens.These cells had a low Ki67 proliferative index and were negative for CD3, CD5, CD10, CD23 and cyclin-D1.
A hepatic biopsy revealed mixed inflammatory infiltration and stage 1 steatohepatitis, with an activity index of 5.
The patient improved clinically and her laboratory parameters normalized after splenectomy.However, 10 months later, the patient presented with weight loss, fever, night sweats, asymmetrical eyelid edema and pleural effusion.At that time, laboratory examinations showed leukocytosis of 30.Restaging CT scans revealed renal infiltration and axillary and abdominal lymph node enlargement.It was concluded that transformation to high-grade B-cell lymphoma had occurred, and treatment with R-CHOP was started, with the following given on day 1 of each cycle: intravenous rituximab, 375 mg/m 2 ; intravenous cyclophosphamide, 750 mg/m 2 ; intravenous doxorubicin, 50 mg/m 2 ; and intravenous vincristine, 1.4 mg/m 2 .In addition, oral prednisone, 100 mg once daily, was given on days 1-5.R-CHOP was administered in combination with intrathecal chemotherapy containing methotrexate (12 mg), cytarabine (60 mg) and dexamethasone (2 mg) (MADIT) and two cycles of methotrexate (3 g/m 2 intravenously) after the last cycle of R-CHOP and radiotherapy to the orbits (24 Gy).Tenofovir (300 mg/day) was started along with the chemotherapy to avoid replication of HBV.
The patient achieved a complete response, and autologous bone marrow transplantation was performed as a consolidation strategy.However, unfortunately, the patient died due to infectious complications during the transplantation.infiltrate involvement, with suspected lymphoid neoplasia.
A hilum splenic lymph node (measuring 2.6 x 1.1 x 1.0 cm) showed lymphoid neoplasia of mature cells with predominance of intermediate to large cells with a nodular pattern.
The diagnosis of splenic diffuse red-pulp small B-cell lymphoma was made and treatment was then instituted, consisting of eight cycles of R-CHOEP: intravenous rituximab (375 mg/m 2 ), intravenous cyclophosphamide (750 mg/m 2 ), intravenous doxorubicin (50 mg/m 2 ), and intravenous vincristine (1.4 mg/m 2 ) on day 1; intravenous etoposide (100 mg/m 2 ) on days 1-3; and oral prednisone (100 mg orally once daily) on days 1-5.It was proposed that this would be followed by autologous stem cell transplantation.After eight cycles of R-CHOEP, the patient achieved complete response, but she became pregnant and chose not to undergo the transplantation.Nonetheless, she continues not to present any evidence of disease today (three years after the treatment).Administration of tenofovir (300 mg/day) was started along with the chemotherapy.

DISCUSSION
We have reported on two patients with chronic non-replicating HBV who developed splenic diffuse red-pulp small B-cell lymphoma.The diagnosis of primary chronic lymphoproliferative disorders of the spleen is established based on analysis on splenectomy tissue, or on integration of clinical data, assessment of lymphocyte morphology in peripheral blood, immunophenotyping of these lymphocytes and evaluation of bone marrow infiltration pattern from biopsy.For our patients, we used both criteria in order to be sure of the diagnosis.In both cases, the splenectomy product demonstrated diffuse neoplastic infiltration consisting of small mature lymphocytes with primary immunophenotype B of the red pulp of the spleen.With this presentation, the diagnosis of splenic marginal-zone lymphoma was ruled out, since this entity is characteristically a primary neoplasm of the splenic white pulp, usually with nodular architectural arrangement.
Other entities that might have formed differential diagnoses were hairy-cell leukemia and hairy-cell leukemia variant.Thus, integration of all these findings, i.e. lymphoproliferative malignancy of small B cells, diffuse infiltration of the spleen and primary from splenic red pulp, with exclusion criteria for "hairy cell" and its variant form, enabled the definitive accurate diagnosis of splenic diffuse red-pulp small B-cell lymphoma in both cases reported here.
We conducted a systematic search in the main electronic databases (PubMed, Google Scholar and Lilacs Library), to find articles relating to splenic diffuse red-pulp small B-cell lymphoma in association with hepatitis B infection.In order to make the search as wide as possible, no limits were applied regarding the date of publication, the language or the research design (Table 1).There were fewer than 100 search results and none of them described any association between splenic diffuse red-pulp small B-cell lymphoma and hepatitis B. 4 consequent to reviewing 85 cases of SMZL.These authors identified four cases with predominance of monomorphic infiltration in the red pulp of the spleen instead of the typical micronodular component in the white pulp commonly seen in SMZL.Later on, two series of cases of splenic diffuse red-pulp small B-cell lymphoma were published by Traverse-Glehen et al. 5 and Kanellis et al. 2 Similarly to our two all cases previously reported were also in stage IV at diagnosis.Diffuse infiltration of the red pulp of the spleen, affecting both cords and sinuses, along with marked DBA-44 positivity, was frequently found by Kanellis et al. 2 (88.2% of their cases), as well as in our cases.Additionally, previous studies described situations of lymph node replacement by diffuse neoplastic infiltrate, with preserved sinuses. 2,4e most common finding in the bone marrow biopsies was predominance of intrasinusoidal lymphoid infiltration, sometimes associated with interstitial and nodular involvement, along with hematopoietic tissue and absent or mild fibrosis.The malignant cells presented with round to slightly irregular nuclei, vesicular chromatin and moderate amounts of pale cytoplasm and cytoplasmic projections similar to SMZL villous cells.These cells expressed CD20, DBA44 and IgG.Presence of annexin A1, CD25, CD3, CD5, CD23, CD103, CD123, CD11c, CD38, CD10, Bcl6, bcl2, cyclin D1 and IgD was uncommon. 1,4Our cases are consistent with those presented in the literature regarding the pathological and immunophenotypic findings of splenic diffuse red-pulp small B-cell lymphoma.The majority of the cases reported in the literature had a normal karyotype and the most frequent chromosomal abnormalities were 7q and 3q deletions and trisomy 18. 1,6 One of our cases showed a complex karyotype.Kanellis et al. 2 found p53 inactivation in all cases of splenic diffuse red-pulp small B-cell lymphoma, comprising either p53 mutation (2/4 cases) or anomalous p53 staining.Mollejo et al. 4 reported that TP53 abnormality was present in 2 out of 13 cases (15.3%): one of these patients showed disease progression and ultimately died of the disease.In the same way as described by these authors, one of our patients also had a p53 deletion and evolved to aggressive disease.Splenic diffuse red-pulp small B-cell lymphoma is an indolent lymphoma and patients can be maintained using a watchful waiting approach or may undergo splenectomy.

Mollejo et al. first proposed this lymphoma as a new subtype in 2002,
Compared with SMZL, splenic diffuse red-pulp small B-cell lymphoma has demonstrated better disease-free survival, but overall survival is not statistically different.At diagnosis, our patients presented with anemia, which is associated with worse prognosis according to Kanellis et al., 2 and both of them had HBV infection as a comorbidity.HBV infection may be associated with worse prognosis, since both patients evolved with aggressive disease.
Virus-induced carcinogenesis is known to occur in several lymphoid malignancies in which the virus has a pathogenic role, such as in Burkitt's lymphoma with Epstein-Barr virus (EBV), adult T-cell leukemia with human T-cell lymphotropic virus-1, and primary effusion B-cell lymphoma with human herpes virus 8. Some malignancies may be caused by chronic stimulation such as in mucosa-associated lymphoid tissue lymphoma, which is associated with Helicobacter pylori, or through an indirect mechanism.While EBV has a direct carcinogenic effect on Burkitt's lymphoma through activating the c-Myc oncoprotein, HIV acts on the immune system to reduce immune surveillance. 7,8V is a small DNA virus that is a member of the Hepadnaviridae family.It replicates through a RNA intermediary and can integrate into the host genome.HBV infection is highly prevalent worldwide, with around 350 million chronically infected individuals.It is endemic in Asia, Africa, the Middle East, Eastern Europe and South America. 9There are 500,000 to 1.2 million deaths due to chronic hepatitis B annually, out of a total of 350 million cases worldwide, 8 and about 340,000 cases of liver cancer relating to HBV. 10 HBV and HCV are known to induce acute and chronic hepatitis and are strongly associated with hepatocellular carcinoma (HCC).HBV carriers have a 200 times higher risk of HCC, and this is one of the highest risks for a human malignancy. 11s pathogenesis involves multiple pathways, including oxidative stress, hepatic inflammation leading to genetic damage and integration of HBV DNA into the host genome, thereby leading to genetic alterations such as chromosomal and gene translocations
18 x 10 9 /l; 22% of the cells were of moderate size and presented loose chromatin and evident peripheral nucleoli, which were morphologically suggestive of centroblasts.Her laboratory values showed hypercalcemia and elevated lactate dehydrogenase (LDH), of more than 3,000 U/l (normal values range from 240 to 480 U/l).Pleural fluid showed infiltration by neoplastic cells.New immunophenotyping of peripheral blood cells revealed aberrant lymphoid cells expressing B antigens, including CD19, surface IgM (sIgM), strong CD20 expression, heterogeneous partial CD79b expression and FMC7 with a lack of CD5 antigen.Bone marrow karyotyping revealed 46,XX [20].The bone marrow biopsy was hypercellular due to infiltration by B cells.Brain and orbit magnetic resonance imaging (MRI) showed infiltration of the optic nerve, bilateral lacrimal glands and CNS.

Case 2 AFigure 2 .
Figure 2. Diffuse infiltrate of small B-cell lymphocytes in the red pulp of the spleen (hematoxylin and eosin, x 200).
However, the clinical data in association with morphological and immunophenotyping data made it possible to safely rule out diagnoses of hairy-cell leukemia (absence of pancytopenia, no cells with the classical morphology of "hairy cell" and immunophenotyping not characterized by strong expression of CD11c, CD25, CD103 and CD123 antigens) or hairy-cell leukemia variant (no lymphocytes with morphology characterized by the presence of central vesicular nucleolus, cytoplasm similar to hairy cell and absence of strong expression of CD11c and CD20 antigens).

Table 1 .
Systematic search of the literature performed on April 4, 2016