Cases of FL are generally classified into 1 of 3 grades based on the guidelines of the World Health Organization (WHO) (23). The grading of FL is based on the average number of centroblasts in 10 randomly selected microscopic x40 high power fields (HPFs). In grade 1 FL, up to 5 centroblasts per HPF are present; grade 2 FL has 6 to 15 centroblasts per HPF, and grade 3 FL has >15 centroblasts per HPF. Grade 3 is further subdivided into 3A, in which some centrocytes are present, and 3B with no centrocytes (24). Approximately 25% of FL patients are in grade 1, 25-50% in grade 2, and ≥50% in grade 3 (25). FL grades 1 and 2 are considered low grade and affected patients do not necessarily require chemotherapy. For grade 3 FL, chemotherapy is often prescribed (24). Approximately 10% of cases of low-grade FL lack t(14;18). However, approximately 30-40 and 70-85% of grades 3A and 3B FL, respectively, are t(14;18)-negative (26,27).

Unlike grade 3A FL, grade 3B FL is less often found to be BCL2-positive by immunostaining and is associated with an increased p53 and MUM1/IRF expression (28). Grade 3B FL is positive for CD10, also known as common acute lymphocytic leukemia antigen (CALLA), in approximately 50% of cases (29), and is associated with translocations in the 3q27 locus, which affects BCL6 expression (30). In these respects, grade 3B FL more closely resembles DLBCL than the other grades of FL (3,26,27,30,31).

Vaandrager et al (32) used a FISH assay to detect sequences flanking the BCL2 gene and found that the absence of t(14;18) was more common in FL grades 2 and 3. Swerdlow et al (23) also found that a lack of BCL2 overexpression was particularly common in grade 3 FL. Grade 3B FLs are composed entirely of centroblasts and are typically associated with a lack of t(14;18) (5,27,30). However, results on the frequencies of different grades in t(14;18)-negative FL have been somewhat contradictory. Perhaps owing to small sample sizes, studies, such as those by Weinberg et al (33) and Horsman et al (21) have found that t(14;18)-negative FLs were more likely to be grades 1 and 2 than grade 3.

The t(14;18) translocation leads to the overexpression of BCL2. However, in t(14;18)-negative FL, BCL2 overexpression is more variable (22,32,34-37). Leich et al (34,35) studied nodal FL grades 1, 2 and 3A, and found that t(14;18)-positive and t(14;18)-negative FL were morphologically indistinguishable. A small subset of t(14;18)-negative FL samples exhibit evidence of BCL2 overexpression, indicating that factors other than the t(14;18) translocation regulate its expression. The majority of t(14;18)-negative FL cases in this previous study (34) expressed BCL6, CD10, and IRF8. In the study by Horsman et al (21) 49 t(14;18)-negative FL cases were analyzed by immunohistochemistry. These researchers found that BCL2 was expressed in 33% of cases. In t(14;18)-negative FLs without BCL2 expression, Leich et al (35) performed microRNA expression analysis and found a decreased expression of T cell leukemia/lymphoma 1A (TCL1A), supporting the hypothesis that this subtype of FL has a late germinal center B cell phenotype.

In addition to BCL2, BCL6 is also frequently expressed in FL (37). While an absence of BCL2 overexpression is a consistent finding in t(14;18)-negative FL, BCL6 expression often accompanies the lack of BCL2 expression (38). In the study by Horsman et al (21), only 33% of 49 t(14;18)-negative FL cases expressed BCL2; however, in the majority of cases, a t(3;14) translocation was present and BCL6 was overexpressed. Jardin et al (39) examined a group of patients with t(14;18)-negative FL who had a 3q27/BCL6 gene rearrangement. Tumor cells in that study were generally negative for CD10, BCL2, CD5, CD23 and CD43, and positive for CD20 and BCL6. In that sample, follicles were significantly larger than those found in t(14;18)-positive FL. Leich et al also found that their sample of grades 1-3A t(14;18)-negative FL lacked CD10 expression (34). Jardin et al (39) speculated that an anti-apoptotic function of BCL6 may act as a surrogate for BCL2 in this group. Gollub et al (40) also found that their samples of t(14;18)-negative FLs with BCL6 translocations were more common in higher grade FL. A total of 60% of their sample of 5 t(14;18)-negative FLs with BCL6 translocations were positive for CD10. However, 100% of their sample of t(14;18)-positive FLs with BCL6 translocations also expressed CD10. In the study by Gollub et al (40), t(14;18)-negative FLs with BCL6 translocations involved the bone marrow less often and did not infiltrate the lymph node capsule as often when compared to t(14;18)-positive FLs. BCL6 translocations in that study affected the phenotype only when they were not accompanied by BCL2 translocations.

Gu et al (19) also found that BCL6 rearrangement was more common in t(14;18)-negative FL than in t(14;18)-positive FL and that BCL6 rearrangements were present in all grades of FL, but were more common in FL grade 3. Pan et al (41) also found that the BCL6 translocation was related to high-grade FL. These studies indicate that BCL6 rearrangement may play a similar role to BCL2 rearrangement in cases where that does not occur. A summary of genes mutated or abnormally expressed in t(14;18)-negative FL is shown in Table II.

An atypical form of FL lacking the BCL2 rearrangement that differs from other examples of t(14;18)-negative FL was reported by Karube et al (42) This FL subtype exhibited an increased expression of IRF4/MUM1, a protein involved in plasma cell differentiation. The samples were also negative for CD10. Patients with CD10^-MUM1⁺ FL were likely to be more elderly than typical FL patients, and more often had grade 3A or 3B morphology and diffuse proliferation. Additionally, 88% of these FLs exhibited BCL6 translocation or amplification.

In a study on the molecular features of t(14;18)-negative FL, Gagyi et al (43) found that t(14;18)-negative FL exhibited no difference when compared to t(14;18)-positive FL in the presence of somatic hypermutations of IGHV genes and the levels of expression of activation-induced cytidine deaminase (AID). This supported a germinal center origin and differentiation in both groups of FL. Additionally, there were no differences in somatic hypermutations of the MYC, RhoH, or PAX5 genes between the 2 groups. These results led Gagyi et al to the conclusion that t(14;18)-positive and t(14;18)-negative should be considered as a single entity. The authors state that, in some cases of t(14;18)-negative FL, BCL2 can be overexpressed by genetic events other than translocation, and other cases of t(14;18)-negative FL, rearrangement of the BCL6 gene could provide the basis of substitution for the lack of BCL2 expression (43).

Another distinct form of t(14;18)-negative FL is pediatric-type FL (44-47). This form of FL is characterized by highly proliferative follicles, which often have blastoid follicular center cells instead of classic centroblasts (44). These cases are negative for BCL2, BCL6, and MYC rearrangements (45). In this form of FL, dissemination beyond the skin is uncommon, and the cases usually have a good clinical prognosis (46). Although previously associated with a primarily pediatric population, there are occurrences in adults (44,47). The study by Martin-Guerrero et al (45) demonstrated that mutations of tumor necrosis factor receptor superfamily 14 (TNFRSF14) accompanied by copy-number neutral loss of heterozygosity (CNN-LOH) at the 1p36 site were more common in pediatric-type FL than in a comparison group with non-pediatric t(14;18)-negative FL. Thus, genetic alterations at the 1p36 site may underlie pathogenesis in pediatric-type FL. The variations in symptomology and gene rearrangements within the t(14;18)-negative FL patient population underscores the diversity of the disease and difficulties related to a coherent classification. These issues suggest that better diagnostic methods are warranted.

Various treatment regimens exist for FL, and the appropriate treatment strategy depends upon the grade and clinical characteristics of the disease. A clinical staging system for lymphomas was published by the Committee on Hodgkin's Disease Staging Classification (51). In this system, stage I indicates the involvement of a single lymph node region or a single extranodal site in the cancer. Stage II indicates the involvement of two or more separate lymph node regions on the same side of the diaphragm. Stage III indicates the involvement of lymph node regions on both sides of the diaphragm. Stage IV indicates diffuse or disseminated involvement of one or more extralymphatic organs. Stage IV also includes localized involvement of liver, bone marrow, or nodular involvement of the lungs.

Approximately 26-33% of FL patients are in stage I or II (52). Approximately 20% of patients with FL undergo spontaneous remission without any treatment (53). In approximately 30-40% of patients with FL, a histological transformation to DLBCL occurs, and the majority patients with histological transformation succumb to the disease within a year (54). In terms of histological transformation and the overall survival of patients, there are no reliably documented differences between patients with t(14;18)-positive and t(14;18)-negative FL (55). As no reliable correlation between BCL2 gene rearrangement status and clinical outcome has been reported, the t(14;18) translocation has not been used as a prognostic marker (56). Furthermore, the results of numerous studies have suggested that a similar set of morphologic, immunophenotypic, and clinical characteristics develop in FL regardless of the presence or absence of a BCL2 rearrangement in FL grades 1-3A. Thus, the generally accepted opinion of these grades of FL is that they constitute a homogenous group with some distinct but many common molecular pathways (43). A depiction of the major targets of drug treatments in t(14;18)-positive and t(14;18)-negative FL is illustrated in Fig. 2. Furthermore, a summary of patient groups with t(14;18)-negative FL with their treatments and outcomes is illustrated in Table III.

Unless the disease can be cured by surgery or local irradiation, FL is generally managed with radiation or chemotherapy. For those projected for treatment with radiation or chemotherapy, clinical monitoring using a 'watch and wait' approach is likely to be recommended until the patient requires treatment (1). For patients who have stage I or II low-grade FL, radiation is usually recommended as a first treatment approach. For patients with low-grade FL with a high tumor burden, chemotherapy is often followed by involved field irradiation therapy (IFRT) (22). In stage III or IV FL, patients typically receive chemotherapy regimens, such as alkylating agents for inhibition of cell growth, purine analogs and anthracycline-based agents (22). Monochemotherapy with chlorambucil or cyclophosphamide is a common approach (22). Treatment with chemotherapy plus rituximab is the accepted standard for most patients and this method is associated with increases in the overall survival of patients (2). Among anthracycline-based regimens, one of the most common treatments is R-CHOP, a combination of 5 drugs: Rituximab, cyclophosphamide (Cytoxan), doxorubicin (Adriamycin), vincristine and prednisone (57). Another commonly used combination chemotherapy is R-CVP, which includes rituximab, cyclophosphamide, vincristine and prednisone (58). CHOP-RIT, the administration of tositumomab/iodine-131 radioimmunotherapy (targets CD20) immediately following CHOP, is also common (59). Another commonly used approach is bendamustine (an alkylating agent) plus rituximab (60). However, rituximab, a monoclonal anti-CD20 antibody, is ineffective in variants of FL that are negative for CD20 (61,62). A recently developed approach is ABT199, a BH3 mimetic investigational drug (venetoclax) that targets the BCL2 pathway (63). However, as the majority of cases of t(14;18)-negative FL lack BCL2 overexpression, this approach would be contraindicated for these patients. A better understanding of the underlying genetics of each variation on the disease may lead to more precisely targeted treatment regimens.

Although the exact mechanisms of rituximab, the most common drug treatment for FL, are not yet fully known (64), it is considered that rituximab achieves its clinical effect by altering intracellular signaling pathways (64). Evidence indicates that rituximab operates by reducing activity of the p38MAPK pathway. This reduction in activity results in the inhibition of the interleukin-10 paracrine cytokine auto-regulatory loop, which in turn inhibits signal transducer and activator of transcription 3 (STAT-3) activity. This inhibition of STAT-3 activity leads to a downregulation of BCL2 expression, and subsequent chemosensitization (65). Additionally, rituximab upregulates expression of Raf-1 kinase inhibitor protein (RKIP) (64). RKIP acts to reduce the activity of the extracellular signal-related kinase (ERK) 1/2 and the NF-κB pathways, which in turn inhibits transcription of AP-1 and NF-κB. The downregulation of both of these pathways leads to the subsequent reduction of BCL2 transcription and expression and susceptibility to apoptosis (64). A better understanding of the effects of drugs on molecular pathways will allow better targeted treatments.

The research group of Leich et al (34,35,66) conducted studies examining clinical differences between patients with t(14;18)-negative and t(14;18)-positive FL. In one study comparing the 2 groups, t(14;18)-negative patients were more likely to have lower stage FL. However, no differences were observed in the other clinical variables of sex, involvement of extranodal sites, B symptoms, lactate dehydrogenase and the Eastern Cooperative Oncology Group (ECOG) performance status (34,67). In another study examining 540 advanced stage and 116 early stage patients with FL treated with chemotherapy and radiation, no differences were observed in patient characteristics between FL cases with and without t(14;18). The survival times patients of patients with advanced-stage FL also did not differ between the t(14;18)-positive and t(14;18)-negative patients (66). Larger population studies should better inform treatment strategies by identifying subtle differences between the t(14;18)-negative and t(14;18)-positive FL populations.

Studies have examined the clinical differences between patients with and without BCL2 and BCL6 rearrangements. Although the majority of results underscore a similarity in clinical presentation between patients with and without BCL2 and BCL6 rearrangements, some differences have been noted. A total of 98 patients with FL and 93 DLBCL were examined by in the study by Watanabe et al (68) with the aim of determining the frequencies of BCL6 and BCL2 translocations in the two patient groups. BCL2 translocations were detected in 77 patients with FL and 22 patients with DLBCL. BCL6 translocations were detected in 14 patients with FL and 14 patients with DLBCL. The patients were followed-up for 29 months after undergoing R-CHOP therapy. For patients with FL and DLBCL, the presence of BCL2 or BCL6 translocation was uncorrelated with clinical outcomes after treatment with R-CHOP therapy. Furthermore, neither overall survival nor progression free survival differed between the patient groups (68).

Some clinical differences between t(14;18)-negative FL patients with BCL6 rearrangements and t(14;18)-positive FL patients have been noted (39). In one study, Jardin et al (39) examined a group of 15 t(14;18)-negative FL patients with BCL6 gene rearrangements and a t(14;18)-positive comparison group. A majority of the t(14;18)-negative group had a t(3;14) translocation. There were no significant differences in age, sex, performance status, bone marrow involvement, or overall survival in comparison to a t(14;18)-positive group. However, in the t(14;18)-negative group, patients were more likely to have a stage III or IV disease. Furthermore, the lymphomas in the t(14;18)-negative group had a prominent nodal architecture, and usually had significantly larger follicles than the t(14;18)-positive FLs. The authors (39) concluded from this that BCL6 may play a role in follicular structure. These results indicated that t(14;18)-negative FL with a BCL6 translocation may constitute a distinct subtype of FL.

Studies have identified uncommon forms of t(14;18)-negative FL, some with diffuse, extranodal expression, and others with expression limited to the skin. One study demonstrated that a subtype of CD10⁻MUM1⁺ FL exhibited distinctive biological and clinical features compared with the more typical CD10⁺MUM1⁻ FL (42). Twenty-two mostly t(14;18)-negative FL patients who were negative for expression of CD10 and positive for expression of MUM1 were generally older than typical FL patients (mean of 67 vs. 58.7 years old). CD10^-MUM1⁺ FL patients also exhibited diffuse FL proliferation and were generally of a higher disease grade, either FL 3A or 3B, having a relatively poor prognosis.

In another study, a different subtype of t(14;18)-negative nodal follicular non-Hodgkin lymphoma exhibited a predominantly diffuse pattern and was characterized by deletions in the chromosomal region 1p36. These patients were more frequently diagnosed as low-grade (stage I and II) FL and generally presented with isolated large tumors (median size of 5 cm) that were frequently localized to the inguinal region. Although a complete remission could be achieved by treatment with radiotherapy alone or chemotherapy in combination with radiotherapy, disease progression generally followed an indolent course (5). This diffuse form of t(14;18)-negative FL represents a previously undocumented form of FL with distinct molecular and clinical features (5).

Moreover, a subtype of generally t(14;18)-negative FL which arises primarily in the skin has also been documented (69). Each of the evaluated 47 patients with FL was in clinical stage I. Compared with t(14;18)-positive cases, these FL cases were less likely to express BCL2 or CD10. These cases also had a higher proportion of extranodal FL sites, and had a significantly higher overall and disease specific five-year survival rate. This subtype of FL has a generally more favorable clinical outcome when compared to t(14;18)-positive FL (19,69).

An unusual case, difficult to diagnose, was reported by Wong et al (70). The authors reported a case of a 51-year-old male with t(14;18)-negative FL who lacked BCL2 expression. The patient was classified as having high-grade 3A FL with areas of large cell transformation within the same lymph node. Neoplastic cells from the patient were positive for CD20, CD79a and BCL6, and were negative for BCL2 and CD10. The clinical diagnosis was FL 3A with diffuse large B cell transformation.

Another report of atypical clinical cases with difficult diagnoses was made by Nybakken et al (71). These researchers reported on 7 patients (5 females ranging in age from 51 to 61 years, and 2 males aged 6 and 61 years). The patients presented with isolated atypical follicles that were composed of cytologically atypical centroblasts located within otherwise normal lymph nodes. The isolated follicles had unusual morphologic features that could be mistaken for lymphoma. The cases were generally positive for CD20 and BCL6, and were negative for BCL2 and CD3. The sites of involvement included peripheral and internal sites, such as a periaortic lymph node and rectal mucosa-associated lymphoid tissue. The authors considered a diagnosis of partial involvement by FL. The cytological characteristics of involved areas were consistent with FL grade 3B (enlarged with vesicular chromatin and prominent nucleoli, and nuclear membrane irregularity inconsistent with the morphology of the surrounding lymph node). However, the pattern of single scattered follicles was not consistent with partial FL. Furthermore, none of the patients had evidence of systematic lymphoma at the time of diagnosis or clinical follow-up visits up to 44 months after diagnosis, and the patients did not require aggressive clinical management. Nybakken et al (71) noted that it is of high importance to differentiate these cases from aggressive FL or a similar disease to avoid unnecessarily aggressive treatment of patients with indolent conditions.

Finally, a surgical treatment for a patient with t(14;18)-negative FL has also been reported (72). The patient was a 74-year-old Japanese female who presented with a complaint of swelling in the submandibular region. The patient was diagnosed with stage I FL. A surgery was performed and the lymph node was removed successfully. Flow cytometric analysis revealed that the lymphoma cells were positive for CD19, CD20, CD23, CD10 and BCL6. The cells were negative for BCL2 and MUM1. There was no recurrence of FL upon follow-up a year post-surgery. This case suggests a distinct type of surgical treatment for certain types of BCL2-negative FL (72).