Large B-cell Lymphoma with IRF4 rearrangement: a pitfall in the pathological diagnosis of lymphoma

Background Large B-cell lymphoma (LBCL) with interferon regulatory factor 4 (IRF4) rearrangement (IRF4+LBCL) is a rare and newly discovered subtype of mature B cell neoplasms. Case presentation Here, we describe a patient of 32 years old who was diagnosed IRF4+LBCL. Histological examination showed the normal structure of the lymphoid tissues were destroyed, and slightly crowded follicular or nodal structures instead. There were obvious necrosis on the surface of tonsil and the central part of some follicles. The monomorphic atypical lymphoid cells proliferated and grew consistently, which were of medium size or large, and the nuclear chromatin was opening. Some tumor cells can be seen around the normal striated muscle tissues near the tonsils. Immunohistochemistry (IHC) could show that CD20, CD79a, MUM-1 and BCL6 were positive, Ki-67 was 80%; CD3, CD5, CD10, BCL2, CD30, CD56, CD99, CD38, and CD138 were negative. In situ hybridization (ISH) of EBER was negative. Fluorescence in situ hybridization (FISH) conrmed that IRF4 gene rearrangement was found in tumor cells. The patient was followed up for 18 months without tumor after chemotherapy. And IRF4+LBCL can be similar to both and diffuse large B-cell lymphoma (DLBCL). But actually this disease is indolent and signicantly different.


Introduction
As a special type of mature B-cell tumor, IRF4 + LBCL is rare, accounting for 0.05% of LBCL [1]. In terms of immunophenotypic, the atypical tumor cells have mature B cell phenotype, what CD20, CD79a and PAX 5 are positive. The most characteristic is the cells strong express IRF4/MUM1 [1,2,3,4]. Usually, Bcl 6 is positive, but CD10 and BCL2 are expressed in different degrees. The proliferation index is usually very high, and the expression pattern of Ki-67 shows the neoplastic follicles lack the polarity of normal lymphoid follicles or reactive proliferating follicles. In most IRF4 + LBCL, the tumor cells showed a complex pattern of genetic change, and most cases were found to have the origin of germatogenic center B cells, including the loss of TP 53 in a group of age-independent patients [1,3,4,5]. And IgH with IRF4 gene translocation can be detected [1,5]. Light chains are rarely involved in translocation. BCL 6 point breakpoints can be seen in some cases. However, almost all cases lacked MYC and BCL2 rearrangement [4,6,7]. Patients had good curative effect after combined immunochemotherapy, that was different from Paediatric-type follicular lymphoma, which could have a good prognosis without radiotherapy or chemotherapy.

Materials And Methods
The specimen was xed with 10% neutral formalin solution, conventional dehydration, transparency, para n embedding, sliced thickness 4 µm, and the dyeing was carried out by using the HE method. In addition, the thin sections were resected 2 µm thick and stained with HE to observe the details of tumor cells. The EnVision method was used in immunohistochemistry. The rst antibodies used were CD20, CD79a, CD3, CD5, CD45RO, CD21, CD23, MUM-1, BCL6, BCL2, CD10, CyclinD1, TDT, CD99, CD38, CD138, Ki-67, purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd and Fuzhou Maixin Biotechnology Co., Ltd. IRF4 isolated breakapark FISH probes was used for gene detection and purchased from ZytoVision Company of Germany.

Clinical history
Male, 32 years old, the tumor is located in the left tonsil. During the onset the patient was snoring occurred more than 20 days ago without obvious inducement, occasionally sore throat, and obvious when swallowing and drinking water. No dysphagia, no itchy pharynx, no cold fever, no nausea and vomiting, no abdominal pain and abdominal distention, no cough and expectoration, no chest tightness and shortness of breath, no dyspnea, no earache, no ear pus. The family members of the patient did not pay attention to this at rst, and he did not receive special treatment. However, the symptom of snoring had not be signi cantly better. Recently, the snoring was aggravated, lasted for a long time and snored louder, so they came to the outpatient department of our hospital to see a doctor. The results showed that the pharyngeal mucosa was ruddy, the soft palatal mucosa was not hypertrophic and relaxed, the uvula was in the middle, no lymphoid follicular hyperplasia was found in the posterior wall of pharynx, the left tonsil was enlarged and the recess was clean.

Pathologic ndings
Under microscope, the tumor was mainly follicular pattern, and a large number of irregular follicles grew by pushing and extruding (Fig. 1). Some of the follicular structures were large and expansive, pushing the surrounding tissues, some of which with a starry sky phenomenon, but no polarity. The mantle zone disappears or becomes narrower and longer. High power, the tumor cells were medium size or large, abundant cytoplasm, irregular nuclei, chromatin vacuole. Basophilic nucleolus can be seen, and also mitotic images. Some tumor cells were looking like centroblasts which have one or more small nucleolus close to the nuclear membrane, and mitosis easy to found (Fig. 2). And also some areas of the tumor grew diffusely and showed a small amount of necrosis. Reactive lymphoid tissues were seen around the tumor.

FISH test result
Most of the tumor cells could be found a single yellow signal appeared in the nuclear of tumor cells, and an orange and a green signal were isolated, what was suggesting IRF4 gene rearrangement (Fig. 5).

Treatment and prognosis
The patients received complete remission after R-CHOP regimen chemotherapy. Follow-up 18 months, so far no recurrence, but need more long-term follow-up data.  [11] analyzed the cohort of WR DLBCL patients with DLBCL primary involved in Waldeyer ring. It found that WR DLBCLs had obvious clinicopathological features compared with conventional DLBCL, often accompanied by localized lesions and follicular growth. It has also been reported that IRF4 + LBCL tumor involves gastrointestinal tract [6, 7,12]. It occurs mainly in children and young people, men are more common, but the reason is unknown. It also found that the prognosis of germatogenic center B cells, including FL and DLBCL, depends to a large extent on age [13][14][15][16]. The prognosis of children group is better than that of adult group. The characteristic change is the strong expression of the immunohistochemical IRF4/MUM1.Gene detection is usually accompanied by rearrangement of IRF4.Although tumor cells are characterized by high heterogeneous nucleus, and the proliferation index is usually high, but their prognosis is good. We must familiarity with its clinicopathological features, and that is helpful for diagnosis and differential diagnosis, avoiding misdiagnosis and causing overtreatment.

Discussion
Our case shows that the patient was a man, the tumor occurred in the left tonsil and had a good prognosis, which is identical to the literatures. Differential diagnosis: 1. paediatric-type follicular lymphomas (PTFL): It mainly involves Waldeyer ring or head and neck lymph nodes, and in the early age group, so it is the diseases that need to be identi ed the most. Pathologically, tumors are follicular growth patterns, and follicles are usually very large, with back-toback growth patterns [6, [17][18][19][20]. The difference is that the tumor follicles of IRF4 + LBCL generally lack creeping morphology, and the positive expression of MUM1 was detected by immunohistochemistry and the abnormality of the IRF4 gene was detected by molecular detection can be identi ed. 2. Burkitt lymphoma: It mainly occurs in children, which is highly invasive lymphomas.

Conclusion
The frequency of IRF4 + LBCL is very high in children, especially in men. In pathological morphology, most of them are follicular growth pattern, but also can be completely diffused growth, and also can have both. The positive expression of IRF4/MUM 1 is constant and strong, and IRF4 rearrangement could be found by gene detection. Although it is a malignant tumor and has a high proliferation index, it has a good prognosis. IRF4 + LBCL is rare, its biological origin and the causes of multiple male children, etc. need more research to explain.

Declarations Compliance with Ethical Standards
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee. Funding:none.

Con ict of Interest
The authors declare that they have no con icts of Interest.

Contributions
Minya Lu performed experiments, analyzed data, and wrote the manuscript; Lisong Teng provided clinical data; Zhe Wang ,Xiaodong Teng and Zhaoming Wang contributed to designed research and analyzed data. Figure 1 HE*100 Irregular follicles grow by pushing and extruding.  Evision*100 Ki67 is about 80%.

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