HIV-related non-Hodgkin lymphomas affecting the oral cavity: a clinicopathologic study of 11 cases

Background HIV-related non-Hodgkin lymphomas of the oral cavity are rare lesions with aggressive clinical behaviour. The aim of this study is to describe the clinicopathological features of a series of HIV-related oral non-Hodgkin lymphomas. Material and Methods Eleven cases of oral lymphomas affecting HIV-positive patients were retrieved from 2012 to 2019. Clinicopathological features regarding age, sex, tumour location, clinical presentation, laboratory findings, disease stage and follow-up were obtained. Histologic, immunohistochemical and in situ hybridization for EBV detection were done for diagnosis confirmation. Overall survival was estimated by Kaplan–Meier curve. Results Males predominated, with a mean age of 40.3 years-old. Maxilla and mandible were the mostly affected. Plasmablastic lymphoma and diffuse large B-cell lymphoma not otherwise specified (NOS) were the main histological types. Lesions presented as reddish ulcerated swellings, representing the first sign of AIDS in six cases. Stage IV were common (7 cases) and the mean HIV viral load was 10,557 copies/mL, with a mean of 266 CD4+ cells/mm3, 1,278 CD8+ cells/mm3 and a CD4+/CD8+ ratio of 0.26. Eight patients died of the disease (72.7%). Overall survival revealed that 78.2% of the patients died after 21 months of follow-up. Conclusions HIV-related oral lymphomas present a poor prognosis usually diagnosed in advanced stages and in our series plasmablastic lymphoma was the most common subtype. Key words:Oral cancer, non-Hodgkin lymphoma, HIV, survival, prognosis.


Introduction
The human immunodeficiency virus (HIV) was initially identified in the 1980s in five young men affected by Pneumocystis carinii pneumonia and other opportunistic infections (1). HIV infection triggers an important immunosuppression condition by disrupting CD4+ T lymphocytes function, leading to a higher risk of developing opportunistic diseases (2,3), especially when CD4+ T cell count falls below than 200 cells/mm3, when the diagnosis of Acquired Immunodeficiency Syndrome (AIDS) is established (3,4). The advent of the Highly Active Antiretroviral Therapy (HAART), led to a significant decrease in the incidence of the most severe systemic conditions associated with AIDS, improving the quality of life of these patients living with HIV/AIDS (3). However, some human cancers are still frequently diagnosed in HIV-positive patients (5), and they are categorized into 2 different types: 1) non-AIDS-defining cancers, including Hodgkin lymphomas, which are associated with advanced age, smoking, race and low CD4+ count (6); and 2) AIDSdefining cancers, usually associated with oncogenic viruses (7). Regarding AIDS-defining cancers, previous studies indicate Kaposi's sarcoma as the most prevalent neoplasm, followed by non-Hodgkin lymphomas (8). Among AIDS-associated NHL, diffuse large B cell lymphoma, primary effusion lymphoma and plasmablastic lymphoma are common subtypes, some of them frequently affecting the oral cavity, whose diagnoses may sometimes represent the clinical manifestation that leads to AIDS/HIV initial diagnosis. Hence, clinicians and pathologists must be aware of the most frequent lymphomas subtypes in the scenario of HIV infection in order to better diagnose and treat these patients. Therefore, the aim of this study is to describe the clinicopathological characteristics and the survival rate of a series of oral lymphomas affecting HIV-positive patients.

Material and Methods
This study was approved by the Ethical Committee of the University Hospital João de Barros Barreto, Belém, Brazil (process no. 4.553.556). All cases diagnosed as oral non-Hodgkin lymphoma between January 2012 and December 2019 were retrospectively retrieved from the pathology files of two Brazilian institutions [João de Barros Barreto University Hospital (Belém), and the Cancer Institute of São Paulo (São Paulo)]. Formalin-fixed, paraffin-embedded tissues were obtained and new histological sections were stained with haematoxylin and eosin (H&E) to be used for diagnosis confirmation by at least two oral pathologists following the current WHO classification of lymphoid neoplasms (9). The cases were analysed and those which were HIVrelated were selected to be included in the final sample. The clinicopathological features were retrieved from the patients' medical files and included age, sex, tumour location, clinical presentation, laboratory findings (CD4+, CD8+, HIV viral load and CD4+/CD8+ ratio), disease stage, treatment, status at last follow-up (dead or alive), and time of follow-up. Overall survival rate was defined as the period from the date of diagnosis to the date of the patient's death or last follow-up. The value ranges of laboratory findings were classified following Taiwo and Hassan (10). Immunohistochemical reactions followed the WHO recommendations to achieve the diagnosis of each case and the most appropriate primary antibody panel was specifically established for each lymphoma subtype. Briefly, reactions were performed in 3µm sections of formalin-fixed, paraffin-embedded tissues that were dewaxed with xylene and then hydrated in a descending ethanol series. Endogenous peroxidase activity was blocked with 10% hydrogen peroxide and antigen retrieval was done using citrate buffer or EDTA solution using a pressure cooker for 3 minutes. After washing in PBS buffer (pH 7.4), the sections were incubated overnight with primary antibodies, and then exposed to high-sensitivity horseradish peroxidase reagents (AD-VANCE; Dako, Carpinteria, CA, USA) and diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich, St Louis, MO, USA). The slides were counterstained with Carazzi haematoxylin for 3 minutes. Positive control histological sections were used for each antibody, while the negative control was acquired by omitting the specific primary antibody. Reactions were descriptively evaluated by at least two oral pathologists. In order to investigate the presence of EBV, all cases were submitted to in situ hybridization (ISH) to detect the virus. A fluorescein-labelled peptide nucleic acid probe (PNA) complementary to 2 nuclear-encoded RNAs (EBER) (Y5200, Dako, Glostrup, Denmark) was hybridized at 55°C for 90 minutes, and then labelling was performed using a PNA ISH detection kit (K5201, Dako). One case of nasal-type extranodal NK/T-cell lymphoma was used as positive control. Carazzi haematoxylin was used for subsequent counterstaining. Cases considered positive for EBV presented a dark blue staining in the nuclei of the tumour cells. The means and percentages are presented as descriptive statistics and overall survival rate was estimated by Kaplan-Meier analysis. The SPSS software version 22.0 was used for statistical analysis.

Discussion
Patients living with HIV/AIDS have a more than 100fold higher risk of developing lymphomas (11), which usually manifest as asymptomatic tumors that may have necrotic regions more frequently in the palate, gingiva and alveolar mucosa (11,12), as demonstrated in our series. Moreover, B-cell origin NHL seems to predominate among patients living with HIV/AIDS and in agreement with previous reports (11,13), we also found PBL to be most common subtype in our sample, although DLBCL predominated in other series (14,15). It is known that CD4 cell count and HIV RNA levels are directly associated with the risk of developing oral NHL (16,17,18). Our results support these findings, since the average CD4 cell count in our case series was low (266 cells/mm3), and HIV RNA was high (10,557 copies/mL). Although, it is unclear how HIV induces chronic B-cell activation with consequent somatic hypermutation, the reduction of immune surveillance allows a permissive effect for EBV to infect B cells, generating chronic B-cell stimulation, which contributes to lymphomagenesis (19). Alternatively, HIV may also produce HIV-derived p17 which promotes changes to the environment (17). Lastly, several soluble cytokines which are potent growth and antiapoptotic factors like IL-6, IL-10, sCD27, sCD44 and sCD30, have been found at elevated levels in HIV-infected individuals (16,17). EBV contributes to the development of B-cell lymphoma by enhancing genetic instability and altering the expression of proto-oncogenes (19,20,21). Hijlkema et al. showed that HIV-infected individuals with EBV DNA loads above 100,000 IU/mL in plasma or serum have increased risk of developing AIDS-related lymphoma.
In agreement with these observations, 8 of the 11 cases in our series were associated with EBV (all PBL and BL cases). EBV inhibits p53 gene expression and activates BCL-2, leading to radiotherapy and chemotherapy resistance through suppression of apoptosis (12,20,22). PBL is an aggressive high-grade B-cell NHL, with a plasmacytic immunophenotype and immunoblastic morphology, commonly described in the oral cavity of HIV-infected individuals, although immunocompetent patients may also be affected (11). Boy et al. (13) and Alli & Meer (11) demonstrated that PBL predominated among oral lymphomas in HIV-positive patients, usually affecting males, as also shown in our series, possibly due to a higher frequency of HIV male patients. Moreover, Castillo et al. (23) reported that HIV-positive PBL patients were younger than HIV-negative subjects and we showed that our patients were under 60 years old, as reported previously (11). In a recent literature review, EBV was observed in 63.4% of PBL cases, being associated with a poor prognosis. In the present series, all PBL patients were EBV-positive and it is hypothesized that HIV infection is responsible for creating conditions in which EBV can infect B cells, preventing their apoptosis (22). At the same time, it is not clear if HIV status interferes PBL prognosis. Despite its aggressiveness, Castillo et al. (23) showed that HIV-negative status is associated with worse overall survival, which could not be demonstrated by other authors. DLBCL has been described as the most common subtype of NHL in HIV positive patients, demonstrating a worse prognosis in the presence of EBV (14,18,24).
Although the presence of this virus is uncommon and we did not find any positive case in our series, Chao et al. (25) (20). CD4+ T cells influence in the survival of B-cell in the germinal centre, and in long-term HIV infection presenting high CD4+ cell count may lead the disruption of germinal centres, preventing BL development (20,21). In addition, activation of c-Myc and inactivation of p53 have also been reported in AIDS-related BL (12). In a Brazilian study, HIV-associated BL more commonly affects male patients in the fourth decade of life, and also show a poor prognosis with a medium survival of 12 months (27). Among NHL affecting the oral cavity, 8-15% are FL. The lesion in currently seen with and indolent course, although transformation to DLBCL may occurs in 20-30% of cases (28). To the best of our knowledge, it is the first case of FL in a people living with HIV/AIDS. EBV is not commonly presented in FL, and when occurs, it is mostly seen in high grade tumours (29).
In conclusion, HIV-related oral lymphomas are uncommon lesions with aggressive biological behaviour. Plasmablastic lymphoma was the most common histological type and mostly affected the maxilla. Due to the association with HIV/AIDS, the present tumours lead to advanced disease stages and poor prognosis.