Clinical analysis of 94 cases of ocular adnexal lymphomas

To investigate the imaging manifestations and pathological features of ocular adnexal lymphoma (OALs) in order to provide relevant information for ophthalmologists. A total of 94 patients with OALs were pathologically confirmed between August 2018 and July 2023, and their demographics, location, subtype, treatment, and prognosis were retrospectively studied. 94 patients with OALs were included in this study, of whom 28 were female (29.79%) and 66 were male (70.21%). The age of the patients ranged from 21 to 80 years, with a predominance of 50–60 years (60/94); monocular onset was common (90/94), and the most site of onset was orbital (74/94), followed by conjunctiva (12/94), eyelids (4/94), vitreous (3/94), and lacrimal gland (1/94). CT and MRI can show the involvement and extent of the lesions, and the pathology of the disease is characterized by mucosa‐associated lymphoid tissue marginal zone B‐cell lymphoma (72/94), diffuse large B‐cell lymphoma (10/94), T‐cell lymphoma (4/94), mantle cell lymphoma (2/94), Burkitt's lymphoma (2/94), small lymphocytic lymphoma (2/94), and follicular lymphoma (1/94). All patients received surgical treatment, 42 patients received local radiation therapy, 35 patients received chemotherapy, 13 patients received radiation therapy and chemotherapy successively, and 3 patients received comprehensive treatment. During the follow‐up, three patients relapsed, four died, one was lost to follow‐up, and the remaining patients had stable conditions. The majority of OALs are mucosa‐associated lymphoid tissue marginal zone B‐cell lymphomas (MALT) with an excellent prognosis. It mostly occurs in people over 50 years of age and most site of the onset is orbital.


| Statistical analysis
SPSS 25.0 (SPSS Inc., IBM, USA) was used for statistical analysis, and the results were expressed as "mean ± standard deviation," and the Kaplan-Meier curve was used for survival analysis, and Log Rank test was used for one-way survival analysis.P < 0.05 was considered to be a statistically significant difference.

| Histomorphological characteristics
Marginal zone B-cell lymphoma (MALT) Microscopic lymphoid hyperplasia, predominantly small lymphoid hyperplasia and mononuclear B-cells, most of which are diffusely distributed, and a small portion of which are nodularly distributed, with a homogeneous morphology, uniformity of size, small cell volume, slightly less cytoplasm, translucency, follicular formation, darkly stained nuclei, rounded or oval shaped, with a slightly irregular nucleus, and an obscure nucleolus, with rare nuclear schizogony.Nuclear schizophrenia is rare, only a few lymphoid follicular growth centers remain; some cells have abundant cytoplasm and are plasma-like differentiated (Figure 4).

Diffuse large B-cell lymphoma
Microscopically, the tumor cells were large B lymphocytes, the tumor cells were diffusely distributed, the cells were medium-sized, with little to medium amount of cytoplasm, some cells had visible nucleoli, the nuclei were rounded, the nucleoli were single or multiple, and the karyorrhexis was easy to be seen, as were the nuclear fragmentation images.

Mantle cell lymphoma
Microscopic tumor cells are diffusely distributed, mainly small and medium-sized lymphoid cells, small to medium-sized cells, dark chromatin, little cytoplasm, irregular nuclei, unclear nucleoli, visible enlarged sets of areas, with the formation of unevenly sized nodules, and visible nuclear schizophrenic images.

Follicular lymphoma
Multiple follicular nodules are seen at low magnification, with thinning of the condylomata, and the cells in the follicles are relatively homogeneous, uniform in size, mostly centrocytic, small or moderately large, and with little phagocytosis.

Burkitt's lymphoma
The tumor cells are moderately large, characterized by a large number of immaturely differentiated lymphocytes with a uniform distribution of macrophages phagocytosing nuclear fragments, and the phenomenon of "starry sky" is seen in some areas.

NK/T-cell lymphoma, nasal type
Diffuse infiltration of medium-sized lymphoid cells with fat necrosis and granuloma-reactive lymphoid cells, massive coagulative necrosis, irregular karyotype, and visible nuclear debris.

| Treatment and prognosis
In this study, all patients diagnosed with lymphoma were treated with surgery.One patient was observed closely observed after surgery.42 patients had received local radiation therapy During the follow-up, three relapsed (3.19%) were MALT lymphomas, four died (4.26%) (one DLBCL, two MCL, and one TCL), one lost to follow-up, the rest maintain a stable condition of their disease.In the overall prognosis of the study, the 5-year OS was 94.6% and the 5-year PFS was 100%.The relationship between patient onset location, age and prognosis were analyzed by the Kaplan-Meier method(-Figure 5).OS for age >50 years and age <50 years were 96.4% and 92.8%, respectively, P = 0.34; all 5-year PFS was 100% with no difference.The 5-year OS of the eyelid or conjunctival or lacrimal gland sites was 92.3% and 95.2%, respectively; the 5-year PFS was 100%, which was not significant.The 5-year OS and 5-year PFS of MALT were both 100%.The 5-year OS and 5-year PFS of DLBCL was 88.9% and 100%, respectively (Figure 6).

| DISCUSSION
OALs are common malignant tumors of the eye, which can involve the orbital and eyelid as well as the conjunctiva, etc.In recent years, with the increasing level of diagnosis, the incidence of OALs has been increasing year by year, with the incidence increasing by 6% to 6.5% per year. 9However, the pathogenesis of OAL is still unclear, and it is multifactorial, related to clonal expansion of lymphocytes induced by persistent antigenic stimulation of chronic inflammation, 22 autoimmune diseases, or pathogenic infections (e.g., EBV, Helicobacter pylori, or Chlamydia psittaci). 23,24In this study, 31 patients underwent in situ hybridization for EBERs, and 4 cases were positive, all of which were TCL, suggesting that TCL is related to EBV infection, and it is hypothesized that EBV infection may be one of the causes of the pathogenesis of TCL, and the relationship between the occurrence of other types of OALs and pathogenic infections still needs to be further investigated.
OALs can occur at any age, and the most common age group of onset is 50-70 years old.The age of onset of the patients in this study was 21-80 (53.05 ± 13.72) years old, and there were 68 patients (72.34%) aged 50-70 years old.In Europe and the United States, the incidence rate was higher in women than in men, 25,26 and this gender difference may be related to the difference in ethnicity or geography.In contrast, in China and other Asian countries, the incidence rate was higher in men than in women, 27,28 which is consistent with our study, where we found that the incidence rate of OALs was 66 cases (70.21%) in men and 28 cases (29.79%) in women.It has been found that OALs usually have monocular onset and can occur in any part of the eye, with the most common site of involvement being the orbit. 10,29,30In this study, 90 cases of monocular onset and 4 cases in Kaplan-Meier curves in patients with marginal zone B-cell lymphomas and diffuse large B-cell lymphoma.
both eyes were found.The site of onset was orbital in 74 cases (78.7%), conjunctiva in 12 instances, and eyelids in 4 cases, the findings were consistent with the conclusion of the literature.The most common pathological type was MALT lymphoma, accounting for 72 cases (76.59%), followed by 10 DLBCL (10.64%) and 4 T-cell lymphomas (4.26%), including 2 nasal types.These findings were consistent with many studies, 17,31 but other types differed slightly, which may be due to the number of cases included in the study.
Orbital CT and MRI are imaging techniques used to detect and diagnose lymphoma within the orbit.CT scans show the lesion's location and structural relationship with the orbital bone, whereas MRI has a higher resolution for soft tissue and is primarily used to detect soft tissue involvement, particularly in the central nervous system.In this study, CT scans revealed soft tissue density shadows with varying morphologies, some of which were isolated nodules, while others were diffuse around the eyeball and adhered to surrounding tissues.
When the extraocular muscles were invaded, extraocular muscle hypertrophy was observed, with indistinct boundaries and significant enhancement.
On the other hand, the MRI scans showed an orbital soft tissue mass, partially surrounding the eyeball, with some cases having diffuse enlargement and involving extraocular muscles.These masses had equal or slightly lower signal in T1WI and slightly higher signal in T2WI.After enhancement, uniform strengthening was observed. 32Overall, orbital MRI is relatively more sensitive than CT in the diagnosis of orbital lymphoma, and most of the patients in this study underwent orbital MRI + enhancement, while only three patients underwent orbital CT due to economic conditions.
T-cell lymphoma can be identified using CD3(+), CD43(+), CD56(+), and LCA(+).It can be challenging to differentiate OAL from orbital lymphoproliferative lesions, orbital inflammatory pseudotumors, and thyroid-related eye diseases.In this study, three patients were diagnosed with malignant lymphoma by pathological histology, but the specimens were too small to conduct immunohistochemical staining.
Ki-67 is a cell proliferation-related antigen, and its proliferation index is an indicator of the activity of tumor cells, and a Ki-67 higher than 30% indicates higher aggressiveness and poorer prognosis. 35In this study, 15 patients had a Ki-67 higher than 30%, and the pathological types were 9 MALT lymphomas (12.5%), 8 DLBCL (80%), 4 TCL (100%), 1 MCL, 2 SLL, and 1 FL, and the follow-up results showed that the prognosis of patients with high ki-67 was relatively poor.The positive rate of Ki-67 in high-grade lymphoma was significantly higher than that of low-grade lymphoma, and Ki-67 in MALT was relatively low (19.76% ± 21.95%).This result suggested that MALT was relatively invasive and low-grade malignancy.However, it has been reported in the literature that MALT lymphoma can be converted into DLBCL. 36In the present study, there were also three cases of MALT with Ki-67 of 60% or more, which may suggest that the tumor cell proliferation of these patients are relatively active, therefore, followup should be strengthened in clinical clinical work.Although Ki-67 is affected by many factors and there are no unified evaluation criteria, the long-term study on the relationship of Ki-67 and OAL prognosis still needs long-term follow-up.Positive clonal rearrangement of the IGH gene indicates that the lesion may contain a small number of malignant cells, and the positive rate of monoclonal rearrangement of the IgH gene in MALT lymphomaswas above 60%. 37In the present study, 40 patients were under went gene rearrangement testing, of which 31 cases were IGH (+), with a positive rate of 77.5%, which is consistent with the above literature report.
The treatment methods for OALs include surgery, radiotherapy, chemotherapy, immunotherapy, or a combination of these methods.
The development of treatment strategies is mainly determined by the type of pathology, the extent of lesion involvement and systemic involvement. 38,39Although, the choice of treatment options for OALs is still controversial, surgery is an important method for the diagnosis and treatment of OALs.Local radiation therapy is more effective, chemotherapy is commonly used in the CHOP regimen, but there will be more adverse reactions, and some believe that radiation therapy is also feasible after chemotherapy is ineffective. 40,41All patients in this Histological subtype is a major predictor of prognosis, and our study found that DLBCL, MCL, and TCL showed a higher mortality rate, which is consistent with the previous study. 42The 5-year OS of MALT was 94.6%, which is similar to the study by Hindsø et al. 43 Similarly, we found that age is a predictor of prognosis, and our study found that age < 50 years had lower OS.In our study, no deaths witnessed among patients with MALT and FL.
There are different types of OALs, each with its own unique characteristics.MALT lymphoma has a slow onset, inert manifestation, less likely to metastasize, high 10-year survival rate, and is very sensitive to radiation therapy, which is recommended to be the first choice of radiation therapy for MALT lymphoma.HE staining of MALT showed infiltration of small to medium-sized lymphocytes in the marginal zone, with few nuclear schizonts and some residual lymphoid follicular structures.Immunohistochemical staining showed CD20(+), CD79a(+), BCL-2 (+), CD10(À), Cyclin D1(À), and a low Ki-67 index.A total of 31 cases (43%) of MALT patients underwent radiotherapy in this study, which achieved better therapeutic effects.DLBCL is common in the elderly, with rapid onset and progression, and is easy to misdiagnose as an inflammatory pseudotumor, with a high degree of aggressiveness.DLBCL Microscopy showed a diffuse infiltration of medium or large B lymphocytes, with many nuclear schizophrenia, immunohistochemical staining CD20(+), CD79a(+), Bcl-2(+), and high Ki-67 index.DLBCL has a lower 5-year OS than MALT, indicating a higher degree of malignancy.The prognosis of postoperative chemotherapy and radiotherapy for DLBCL is similar.MCL is a kind of malignant lymphoma with high degree of aggressiveness, rapid progression, relatively rare, not more than 10% of lymphomas in the ocular appendages. 44FL Microscopy showed proliferating lymphocytes of small to medium sizes with follicle-like morphology.Immunohistochemical staining showed diffuse infiltration of CD20(+) cells, CD10(+), and Ki67 (60%+).T-cell lymphoma has high degree of malignancy, high aggressiveness, poor prognosis, preferably outside the lymph nodes, and cases occurring in the appendages are very rare, and are mainly metastatic from neighboring organ lesions such as the nasal cavity and sinuses to the ocular appendages. 45T-cell lymphoma Microscopy showed diffuse infiltration of medium-sized lymphocytes with irregular nuclei and nuclear fragments, immunohistochemical staining of CD3(+), CD43(+), CD56(+), LCA(+), high Ki-67 index, and molecular pathology results showed EBER(+).
There were several limitations in our research.This was a retrospective study and was conducted only at our institution.Lack of accurate clinical information in some patients may have led to selection bias as most patients did not undergo surgery at our institution.
Therefore, there is a need for future multicenter, prospective studies to improve the credibility and accuracy of the data.Treatment strategies should also be selected according to the type of lymphoma to reduce the incidence of recurrence and systemic spread, and improve the survival rate and quality of life of patients.

AUTHOR CONTRIBUTIONS
Yu-ying Sun performed the research, collected the data, wrote the paper and revised the paper; Shu-ai Luo collected the data and contributed to the analysis; Chun-yu Huang designed and performed the research, provided clinical advice and supervised the report.
This study retrospectively analyzed the clinical data of 94 patients with confirmed OALs who were admitted to the hospital between August 2018 and July 2023.All patients were diagnosed with OALs through pathological examination.All the patients underwent complete excision.The majority of patients underwent complete excision of the lesion and a small number of patients underwent enucleation.General information, clinical examination (imaging and pathology) findings, treatment modalities (surgical treatment, systemic chemotherapy, radiotherapy, or combination therapy) and prognosis (including disease regression, time to recurrence, time to death, and date of last follow-up) were collected and analyzed.OAL pathological categorization standards are based on World Health Organization (WHO) classification and diagnostic criteria for "Neoplastic Diseases of Hematopoietic and Lymphoid Tissues." 20,21Outpatient and telephone follow-up of patients' post-treatment outcomes.

2. 2 |
Pathological analysis Pathological analysis was conducted on all samples using 10% neutral formaldehyde fixation, routine dehydration, transparent paraffin embedding, 4 μm tissue sections, and at least three discontinuous sections were taken for HE staining.Two ologists consulted the pathological sections, and further examination was conducted using immunohistochemical staining and immunoglobulin heavy chain (IGH) gene rearrangement technology.After initial diagnosis based on HE staining, immunohistochemical testing was performed: CD20, CD79a, CD3, CD5, CD23, CD10, Cyclin D1, CD43, CD38, Bcl-2, and Bcl-6 labeling on all specimens predisposed to B-cell lymphoma; CD45RO, CD56, and CD3 markers were performed on specimens predisposed to TCL to assist in the determination of T-cell lymphoma.Ki-67 labeling was performed in all cases to detect the proliferative activity of tumor cells, and Igκ immunoglobulin κ light chain and Igλ immunoglobulin λ light chain were performed in some cases to validate the initial diagnosis and determine the subtype.

1
Distribution of ocular adnexal lymphomas patients in different age groups.

3. 2 |
Imaging examinationAll patients underwent imaging examinations of the orbit, including CT and MRI with contrast, which clearly revealed the location and extent of the lesion.In five cases of orbital CT scanning + enhancement examination, the main manifestations were occupations of the eye, intraorbital soft tissue shadows, with various morphologies, diffuse hyperplasia encircling the eyeball, adherence to adjacent tissues, unclear borders, obvious enhancement, and hypertrophy of extraocular muscles when infringing on the extraocular muscles (Figure2); 89 cases of orbital MRI + enhancement examination, with clear borders in 8 cases, and unclear borders in 81 cases, which could be seen as a mass of flaky or striated soft tissue signal foci or irregular soft tissue signal foci, and the borders with surrounding tissues were not clear, especially when infringing on the ocular muscles, with unclear demarcation.Or irregular soft tissue signal foci, and the border with the surrounding tissue is not clear, especially when the eye muscle is invaded, the demarcation is unclear, the lesion T1WI is equal or slightly low signal, T2WI is slightly high signal, enhancement can be seen uniformly strengthened; nine cases of sieve sinus, maxillary sinus mucous membrane thickening, and the bone of the base of the skull are not damaged (Figure3).
study were underwent surgery, one patient was under close observation after surgery, 42 patients received local radiation therapy, 35 patients received chemotherapy, 13 patients received radiotherapy combined with chemotherapy, and 3 patients received comprehensive treatment.The main purpose of surgery is to clarify the diagnosis and remove the lesion as much as possible while ensuring the structure and function of the ocular tissues.Surgery combined with close follow-up can be used for low-grade limited lesions, while for highgrade or relatively invasive OALs with a large infiltration range, postoperative radiotherapy or chemotherapy should be given, and comprehensive treatment should be performed if necessary.
To summarize, OALs can occur in different parts of the eye, and the prognosis is closely related to the type of lymphoma.Treatment strategies should be rationally selected according to the pathologic type to reduce the incidence of recurrence and systemic spread.In clinical practice, diagnosis should be based on clinical manifestations, imaging examinations, and pathological results to avoid misdiagnosis.
Clinicopathological characteristics of ocular adnexal lymphomas patients by tumor histological subtype.