Establishment Of A Combination Scoring Method For Diagnosis Of Ocular Adnexal Lymphoproliferative Disease

ObjectiveLymphoproliferative disease(LPD) of the ocular adnexa is a relatively common primary ocular adnexal tumor. LPDs of the ocular adnexa encompasse the majority of orbital diseases, and mainly includes reactive follicular hyperplasia(RFH), atypical lymphoid hyperplasia(ALH), and mucosa-associated lymphoid tissue lymphoma(MALToma). It is nearly impossible to categorize all ocular LPDs into clearly defined types, as the histological features in different cases might vary extensively. Currently, due to lack of specific clinical signs and diagnostic immunohistochemical biomarkers, it is difficult for pathologists to distinguish MALToma from ocular RFH and ALH, thus make the clinician embarrassed in chosing clinical management. Lymphoid follicle(LF) usually is observed in the histological examination of LPDs. In a typical LF of peripheral lymphoid tissues, transmembrane immunoglobulin D(Ig D) is strictly expressed in mantle zone(MZ) as an approximately smooth curved contour, whereas CD23~+ follicular dendritic cells(FDCs) exist in the germinal center(GC) as a high-density meshwork. BIOMED-2 Standardized clonality anaslysis system is recognized as an accurate molecular pathological testing technique to help the MALToma diagnosis. Thus, we observed LFs in all three subtypes of LPDs and sought to find the difference among ocular adnexal RFH, ALH, and MALToma, basing on CD23/Ig D immunostaining and Ig H clonal rearrangement. We also analyzed the associations of clinicopathological manifestations among the three lymphoproliferative subtypes in a Chinese population.MethodsWe carried out a retrospective study of 125 patients with primary ocular adnexal LPD who were diagnosed at the Institute of Orbital Disease, General Hospital of Chinese People’s Armed Police Forces(Beijing, China) from January 2006 to September 2014. The study population was divided into three subgroups based on diagnosis: RFH(n = 54), ALH(n = 28), and MALToma(n = 43). We analyzed the clinical features of 125 patients with ocular adnexal LPDs and investigated the structure of LF in paraffin-embedded tissue samples using anti-CD23 and anti-Ig D by immunochemistry(IHC). We combined the expression of CD23/Ig D and the structure of LF to establish a scoring method for differential diagnosis of LPDs. According to the BIOMED-2 protocol, we further detected Ig H gene clonal rearrangement of 73 cases, including 35 RFH, 17 ALH and 21 MALToma. The Ig H gene rearrangements were amplified by multiplex primer polymerase chain reaction(PCR), analyzed by polyacrylamide gel electrophoresis and compared with results of negative controls.Results1. Several trends in clinical characteristics could be distinguished among the three disease types. There was a great variation in patient age(13–93 years), with mean ages of 55.92, 60.44, and 64.95 years(range: 13–82, 23–77, and 35–93 years) in the RFH, ALH and MALToma groups, respectively. The age of patients with RFH was significantly lower than that in the MALToma group(P < 0.001). 28/54 RFH patients(51.85%) were female, whereas 9/43 MALToma patients(20.93%) were female(P = 0.002). The majority of LPDs occurred in the orbit soft tissue, conjunctiva, lachrymal gland, and extraocular muscle. Periorbital swelling and proptosis were the most common clinical symptoms in all patients. Conjunctiva involvement was rarely present in RFH and was more prevalent in ALH and MALToma(P < 0.0167), whereas, lachrymal gland involvement was more prevalent in RFH patients than in the other two groups(P < 0.0167).2. HE staining results revealed LF-like structures and similar pathological features in tissues of the three disease. Variations in LF size, prominent CD23~+ GCs, and intact IgD~+ MZs were observed in tissues from RFH patients. Conversely, the ALH specimens showed scattered, dispersed residual FDC networks and scattered, disrupted IgD~+ MZ fragments. MALToma samples showed a prominent lack of both CD23~+ FDC meshwork and IgD~+ mantle fragments, and few CD23~+ or IgD~+ lymphocytes were observed.3. In all 125 LPD cases, the Pearson’s correlation was significant between the Ig D positive rate and CD23 positive rate(Pearson’s r = 0.729; P < 0.001). The rates of Ig D and CD23 staining in RFH were higher than that in another two groups.4. Based on the integrity of the LF structure and the expression level of CD23/Ig D, we individually established two scoring methods(LF score and expression score) for all samples. The expression score and LF score of RFH were significantly higher than those of ALH or MALToma, whereas no significant differences were observed between ALH and MALToma. Then, we combined the expression score and the LF score together to establish a scoring method for lymphoid follicles. The three types of LPDs were found to be effectively diagnosed using the combination score(P < 0.05/3). The combination score was negatively related with patient age(Pearson’s r =-0.305; P = 0.001). Additionally, we found that the combination score was higher in female patients than in male patients. Furthermore, patients with bilateral involvement had a higher combination score than those with unilateral involvement.5. According to the BIOMED-2 protocol, eighteen of 21 MALToma and eight ALH specimens were detected monoclonal rearrangement, but nine AL H cases and all RFH lesions were negative for clonal rearrangement.The sensitivity and specificity for MALToma samples were 85.7%(18/21). The sensitivity for ALH cases was 35.3%(6/17).ConclusionsSeveral trends in clinical characteristics could be distinguished among the three disease types of ocular adnexal LPDs, including age, sex, disease duration, involved location and laterality. The combined use of Ig D and CD23 immunostaining clearly reveals the LF structure. The scoring system was useful to assess the alterations in amorphous follicle structure to better distinguish RFH, ALH, and MALToma based on the Ig D and CD23 IHC study. The intriguing phenomenon of changes in the follicles among the three groups suggests that the follicular structure becomes gradually disrupted from RFH to MALToma. The scoring method based on morphological evidence might be easy to perform and provides a potential useful method for differential diagnosis of three types of ocular adnexal LPDs. Ig H gene rearrangements technique is sensitive and helpful to evaluate some cases which are difficult in making a final diagnosis.