requiring additional cooperating mutations in specific stem cell programs to be converted into the lymphoma originating cells able to generate and sustain lymphoma development

Follicular lymphoma mainly affects older adults. The average age at diagnosis is about 55. Men and women are nearly equally affected. Follicular lymphoma is a slow-growing disease with minor warning signs that often go unnoticed for a long time before it is diagnosed. The disease is often advanced before a diagnosis is made. Most individuals are diagnosed in stage III or IV. However, even in advanced stages there is no immediate threat to life. The disease has a "waxing and waning" course, meaning that it flares up and regresses a number

The first sign of the condition is often a painless swelling in the neck, armpit or groin that is caused by enlarged lymph nodes. However the majority of patients initially present with disseminated disease that follows a relatively indolent clinical course. Patients with follicular lymphoma typically present with superficial lymph nodes of small to medium

In some patients, the first symptoms are more insidious and related to the slow growth of lymph nodes in deep areas, usually in the infradiaphragmatic territories such as the retroperitoneum, the mesenteric, or the iliac areas. In those cases, patients may complain of atypical symptoms while the tumor bulk can be important, with single or confluent lymph nodes. Primary mediastinal involvement is uncommon, as well as isolated splenic enlargement[13]. Other symptoms may include loss of appetite and tiredness. The general status of the patient is usually preserved, Some people have night sweats, unexplained high temperatures and weight loss. These are known as B symptoms. Others have an altered

Primary involvement of extranodal areas is also very uncommon[14]. The bone marrow is involved in 50% to 60% of the cases. Follicular lymphoma can arise in the gastrointestinal (GI) tract, predominantly in the duodenum or the small intestine, [15,16]. where it can eventually represent the unique site of disease. Lymphoma infiltration may be unifocal or multifocal[13]. The new World Health Organization–European Organization for Research on Treatment of Cancer (WHO-EORTC) cutaneous lymphoma classification recognizes an entity called "primary cutaneous follicle center cell lymphoma" that includes what was previously known as "cutaneous follicular lymphoma variant" in the WHO classification. [17]. Other follicular lymphomas that may be considered as peculiar entities with a distinct behavior are those involving the testis [18] and the rare cases of follicular lymphoma

Follicular lymphoma is characterised by response to treatment with disease-free or asymptomatic disease intervals, alternating with recurrence/progression and may transform to aggressive lymphoma at a rate of around 3% per year [13]. This feature is usually—but not systematically—associated with a poor outcome [20].The clinical factors associated with the risk of transformation (as well as the biology underlying this

size. All common superficial territories can be involved by the disease.

[12].

**Signs and Symptom**

of times over years.

performance status.

encountered in children [19].

**3. Clinical aspects of follicular lymphoma**

Several prognostic parameters for follicular lymphomas were identified in the last two decades, that led to the development of some prognostic indexes. The International Prognostic Index developed for aggressive lymphomas was also found to be able of predicting the outcome for patients with follicular lymphoma, but the proportion of patients in the higher-risk categories was usually limited. [13]

The first prognostic system specific to follicular lymphoma was developed by the Italian Lymphoma Intergroup (ILI) in the late 1990s [22]. Currently, the Follicular Lymphoma International Prognostic Index (FLIPI) [23] is deemed to be more applicable across a range of clinical settings . Both systems were developed prior to the introduction of monoclonal antibody therapy, which has profoundly changed the treatment and outcome of follicular lymphoma [24]. Hence, the FLIPI-2 was recently developed, in a prospective series of patients needing treatment, using parameters which were not previously amenable to retrospective analysis, and may represent a promising new tool for the identification of follicular lymphoma patients with different risk profiles in the era of immunochemotherapy [25].

The description of the Follicular Lymphoma International Prognostic Index (FLIPI) represents an important step in identifying patient subgroups with predictable outcome and comparing the results of clinical trials. Analyses of gene expression profiles or constitutive gene variations may also provide additional insights for prognostication in the near future. Furthermore, these data underline the complex interactions between the tumor cells and their microenvironment; recent attempts to translate these findings with immunohistochemical studies remain unable to robustly predict patient outcome. The therapeutic strategies in follicular lymphoma have been transformed by monoclonal antibodies, used alone or in combination with chemotherapy. Treatment options should be adapted to the clinical features at diagnosis and appear to be able to modify the overall survival of some subgroups of patients. Further efforts may focus on strategies that can alter the natural history of this disease [13].

### **5. Treatment**

There is a general consensus that the natural history of advanced stages of low-grade or indolent lymphoma has not changed for the last 30 years. Patients with advanced stages of indolent non-Hodgkin's lymphoma have been treated for many years with various approaches, including deferred initial therapy (watch and wait), single-agent alkylating agents, radiation therapy, combination chemotherapy, and autologous stem-cell transplantation. Unfortunately, it has been impossible to demonstrate that the long-term prognosis for these patients has significantly changed with any these treatment options [26].

The decision to start first-line treatment depends not only on the stage but also on the symptoms of the disease [27].According to The Swiss Group for Clinical Cancer Research

Stratification of Patients with Follicular Lymphoma 375

Follicular NHLs are characterized by a follicular growth pattern , but a diffuse area may also be present. The follicles of follicular lymphoma were more closely packed together, more

These follicles contained a comparatively monomorphic cellular phenotype, mostly of centrocytes (small cells with cleaved nuclei) and occasional centroblasts (large cells with multilobated nuclei and multiple nucleoli). Mitotic figures were fewer than in reactive follicular hyperplasia (RFH) and tingible body macrophages were sparse or absent. Polykaryocytes were not observed in RFH or follicular lymphoma and sclerosis was only

The distinction of FL from RFH is essential, as the latter represents a benign condition [43].A number of morphological features are of value in making this distinction; in particular, a low density of follicles per unit area, The follicles are separated by wide interfollicular areas with prominent mantle zones. The variably sized follicles were composed of a polymorphic population of lymphocytes, dendritic cells, and tingible body macrophages that imparted a"starry sky" pattern. Mitotic figures were conspicuous. The presence of polarity within follicles and the lack of a monomorphic appearance within the follicles all favor RFH [42].

Being a mature B-cell lymphoma, FL express a large spectrum of B-cell markers, such

FL cells express antigens of the germinal center including CD10 and Bcl-6. Most cases of FL express Bcl-2 protein which is highly correlated with the presence of the t(14;18) but may be

The follicles of RFH and follicular lymphoma stained positively for the B cell marker CD20 which highlight the difference in the width of the interfollicular region between the follicles. In RFH, T cells were predominantly located in the interfollicular zones but also lightly percolated throughout the center of the follicles, demonstrated by T cell marker CD3. CD5 stained T cells are in a nearly identical pattern. T cells stained positively for CD3 and CD5 in follicular lymphoma located within the interfollicular zones. The latter also displaying a

Follicles in both RFH and follicular lymphoma were positive for BCL-6. BCL-2 staining revealed that the follicular centers in RFH were negative and those in follicular lymphoma were positive. CD10 staining was positive for both RFH and follicular lymphoma but

Some cases of both RFH and follicular lymphoma manifested a vague follicular architecture that is difficult to detect. In these cases, CD23, a dendritic cell marker, outlined the follicles of RFH more clearly by disclosing a scaffolding or a cluster of interconnecting cells with elongated cytoplasmic processes. Follicular lymphoma manifested the same pattern of CD23 staining. Ki-67, a marker for cellular proliferation, showed dense positivity that was polarized (unevenly distributed or centered at one edge of the follicle) within RFH follicles. In contrast, follicular lymphoma demonstrated a more diffuse, evenly distributed staining

monotonous in size and shape, and frequently lacked an obvious mantle zone [41].

**6. Cytomorphology of follicular lymphoma**

focally present to a minimal degree [42].

as CD20, CD19, CD22, CD79a, and Pax51 [44].

expressed in cases with a clonal karyotype lacking the t(14;18) [45].

more prominent spillover of CD20-positive cells than in RFH [42].

showed a greater interfollicular staining in follicular lymphoma [41].

**7. Immunophenotype**

(SAKK) trials at least one of the following signs is present in order to start treatment: B symptoms; symptomatic enlarged lymph nodes or spleen; steady, clinically significant progression of lymphadenopathy, splenomegaly or other follicular lymphoma lesions documented by a 50% increase in size over a period of at least 6 months; involvement of at least 3 nodal sites (>3 cm), bulky disease (>7 cm), haemoglobin <10g/dL, and platelets <100 x 109/L due to bone marrow infiltration or splenomegaly [28].

Inspite of encouraging early results [29],there is still no solid data to confirm that early treatment with rituximab significantly delays the need for new therapy,and whether this approach may alter the natural history of the disease and there is also conflicting results Whether early treatment of follicular lymphoma is associated with a decreased risk of transformation [27].

Horning [30],reported the results of sequential treatment studies conducted at Stanford University from 1960 to 1991. Median survival times have ranged from 7 to 10 years, and the OS for each group of studies was overlapping. Indolent lymphoma is generally considered incurable because no plateau in the survival curve has been demonstrable. However, the development of monoclonal antibodies (MoAbs) has revolutionized the treatment of patients with follicular lymphoma [26].

Many trials utilizing different protocols of combination chemotherapy plus rituximab,eg. CVP [31], CHOP [32,33] concluded that OS for patients with follicular lymphoma has improved over time and that the choice of initial therapy may matter. Sacchi et al [34],concluded that FFS and OS have significantly improved in advanced-stage follicular lymphoma patients treated on GISL( Gruppo Italiano Studio Linfomi) protocols during the last 18 years. These improvements are related to evolving front-line and salvage therapies, particularly the introduction of rituximab in combination with chemotherapy

For patients needing therapy, most patients are treated with chemotherapy plus rituximab, which has improved response rates, duration of response, and overall survival. Randomized studies have shown additional benefit for maintenance rituximab both following chemotherapy-rituximab and single agent rituximab [35]. Rituximab maintenance for up to 2 years has a favorable side effect profile and, based on a systematic meta-analysis, substantially prolongs PFS and OS in relapsed disease even after antibody-containing induction in patients who have not received antibody as first-line therapy [36].

Stem cell transplantation (SCT) including both autologous and allogeneic SCT or experimental agent therapy is considered for recurrent disease [35].

In the small proportion of patients with limited non-bulky stages I–II, radiotherapy (involved or extended field, 30–36 Gy) is the preferred treatment having a curative potential [37]. However, In selected cases a watchful waiting may be discussed to avoid the side effects of radiation [38]. Radioimmunotherapy or chlorambucil plus rituximab remains an alternative in patients with low risk profile or contraindications for a more intensive chemoimmunotherapy [39,40].

Therfore, the therapeutic strategies in follicular lymphoma have been transformed by monoclonal antibodies, used alone or in combination with chemotherapy. Treatment options should be adapted to the clinical features at diagnosis and appear to be able to modify the overall survival of some subgroups of patients. Further efforts may focus on strategies that can alter the natural history of this disease [13].

(SAKK) trials at least one of the following signs is present in order to start treatment: B symptoms; symptomatic enlarged lymph nodes or spleen; steady, clinically significant progression of lymphadenopathy, splenomegaly or other follicular lymphoma lesions documented by a 50% increase in size over a period of at least 6 months; involvement of at least 3 nodal sites (>3 cm), bulky disease (>7 cm), haemoglobin <10g/dL, and platelets <100

Inspite of encouraging early results [29],there is still no solid data to confirm that early treatment with rituximab significantly delays the need for new therapy,and whether this approach may alter the natural history of the disease and there is also conflicting results Whether early treatment of follicular lymphoma is associated with a decreased risk of

Horning [30],reported the results of sequential treatment studies conducted at Stanford University from 1960 to 1991. Median survival times have ranged from 7 to 10 years, and the OS for each group of studies was overlapping. Indolent lymphoma is generally considered incurable because no plateau in the survival curve has been demonstrable. However, the development of monoclonal antibodies (MoAbs) has revolutionized the treatment of

Many trials utilizing different protocols of combination chemotherapy plus rituximab,eg. CVP [31], CHOP [32,33] concluded that OS for patients with follicular lymphoma has improved over time and that the choice of initial therapy may matter. Sacchi et al [34],concluded that FFS and OS have significantly improved in advanced-stage follicular lymphoma patients treated on GISL( Gruppo Italiano Studio Linfomi) protocols during the last 18 years. These improvements are related to evolving front-line and salvage therapies,

For patients needing therapy, most patients are treated with chemotherapy plus rituximab, which has improved response rates, duration of response, and overall survival. Randomized studies have shown additional benefit for maintenance rituximab both following chemotherapy-rituximab and single agent rituximab [35]. Rituximab maintenance for up to 2 years has a favorable side effect profile and, based on a systematic meta-analysis, substantially prolongs PFS and OS in relapsed disease even after antibody-containing

Stem cell transplantation (SCT) including both autologous and allogeneic SCT or

In the small proportion of patients with limited non-bulky stages I–II, radiotherapy (involved or extended field, 30–36 Gy) is the preferred treatment having a curative potential [37]. However, In selected cases a watchful waiting may be discussed to avoid the side effects of radiation [38]. Radioimmunotherapy or chlorambucil plus rituximab remains an alternative in patients with low risk profile or contraindications for a more intensive

Therfore, the therapeutic strategies in follicular lymphoma have been transformed by monoclonal antibodies, used alone or in combination with chemotherapy. Treatment options should be adapted to the clinical features at diagnosis and appear to be able to modify the overall survival of some subgroups of patients. Further efforts may focus on

particularly the introduction of rituximab in combination with chemotherapy

induction in patients who have not received antibody as first-line therapy [36].

experimental agent therapy is considered for recurrent disease [35].

strategies that can alter the natural history of this disease [13].

x 109/L due to bone marrow infiltration or splenomegaly [28].

transformation [27].

patients with follicular lymphoma [26].

chemoimmunotherapy [39,40].

### **6. Cytomorphology of follicular lymphoma**

Follicular NHLs are characterized by a follicular growth pattern , but a diffuse area may also be present. The follicles of follicular lymphoma were more closely packed together, more monotonous in size and shape, and frequently lacked an obvious mantle zone [41].

These follicles contained a comparatively monomorphic cellular phenotype, mostly of centrocytes (small cells with cleaved nuclei) and occasional centroblasts (large cells with multilobated nuclei and multiple nucleoli). Mitotic figures were fewer than in reactive follicular hyperplasia (RFH) and tingible body macrophages were sparse or absent. Polykaryocytes were not observed in RFH or follicular lymphoma and sclerosis was only focally present to a minimal degree [42].

The distinction of FL from RFH is essential, as the latter represents a benign condition [43].A number of morphological features are of value in making this distinction; in particular, a low density of follicles per unit area, The follicles are separated by wide interfollicular areas with prominent mantle zones. The variably sized follicles were composed of a polymorphic population of lymphocytes, dendritic cells, and tingible body macrophages that imparted a"starry sky" pattern. Mitotic figures were conspicuous. The presence of polarity within follicles and the lack of a monomorphic appearance within the follicles all favor RFH [42].

### **7. Immunophenotype**

Being a mature B-cell lymphoma, FL express a large spectrum of B-cell markers, such as CD20, CD19, CD22, CD79a, and Pax51 [44].

FL cells express antigens of the germinal center including CD10 and Bcl-6. Most cases of FL express Bcl-2 protein which is highly correlated with the presence of the t(14;18) but may be expressed in cases with a clonal karyotype lacking the t(14;18) [45].

The follicles of RFH and follicular lymphoma stained positively for the B cell marker CD20 which highlight the difference in the width of the interfollicular region between the follicles. In RFH, T cells were predominantly located in the interfollicular zones but also lightly percolated throughout the center of the follicles, demonstrated by T cell marker CD3. CD5 stained T cells are in a nearly identical pattern. T cells stained positively for CD3 and CD5 in follicular lymphoma located within the interfollicular zones. The latter also displaying a more prominent spillover of CD20-positive cells than in RFH [42].

Follicles in both RFH and follicular lymphoma were positive for BCL-6. BCL-2 staining revealed that the follicular centers in RFH were negative and those in follicular lymphoma were positive. CD10 staining was positive for both RFH and follicular lymphoma but showed a greater interfollicular staining in follicular lymphoma [41].

Some cases of both RFH and follicular lymphoma manifested a vague follicular architecture that is difficult to detect. In these cases, CD23, a dendritic cell marker, outlined the follicles of RFH more clearly by disclosing a scaffolding or a cluster of interconnecting cells with elongated cytoplasmic processes. Follicular lymphoma manifested the same pattern of CD23 staining. Ki-67, a marker for cellular proliferation, showed dense positivity that was polarized (unevenly distributed or centered at one edge of the follicle) within RFH follicles. In contrast, follicular lymphoma demonstrated a more diffuse, evenly distributed staining

Stratification of Patients with Follicular Lymphoma 377

cells with mitotic figures and tingible body macrophages (arrow). (Top right) Hematoxylineosin-stainedsection of follicular lymphoma shows mostly small centrocytes with cleaved nuclei and an occasional centroblast (arrow) with a large nucleus and peripheral nucleoli. Inset shows the closer, less well-defined arrangement of several follicles. (Middle left) CD20 stains B cells within the follicles of RLH. The inset indicates the substantial width of the interfollicular zones. (Middle right) CD20 also stains the B cells in the follicles of follicular

inset.(Bottom left) CD3 stains T cells in the interfollicular zones (inset); the T cells can also be

lymphoma; the close arrangement of the follicles can again be appreciated in the

diaminobenzidine chromogen, 200, insets 25) [42].

seen percolating throughout the folliclesof RLH. (Bottom right) Follicular lymphoma manifests nearly identical CD3 staining, with CD3\_ cells prominent in the interfollicular zones (inset) as well as scattered within the follicles. (Top left and top right, hematoxylineosin, 400, insets, 25;Middle left through Bottom right, immunoperoxidase reactions,

pattern within the follicular centers. Polyclonality was evident in RFH, with in situ hybridization revealing roughly equal populations of both kappa and lambda light chains. In follicular lymphoma rare cells stained positive for kappa or lambda [42].

Fig. 1. Histopathological and immunohistochemical characterization of reactive lymphoid hyperplasia and follicular lymphoma.(Top left) Hematoxylin-eosin staining demonstrates follicles in reactive lymphoid hyperplasia (RLH) (inset). The follicles are composed of small

pattern within the follicular centers. Polyclonality was evident in RFH, with in situ hybridization revealing roughly equal populations of both kappa and lambda light chains. In follicular lymphoma rare cells stained positive for kappa or lambda [42].

Fig. 1. Histopathological and immunohistochemical characterization of reactive lymphoid hyperplasia and follicular lymphoma.(Top left) Hematoxylin-eosin staining demonstrates follicles in reactive lymphoid hyperplasia (RLH) (inset). The follicles are composed of small cells with mitotic figures and tingible body macrophages (arrow). (Top right) Hematoxylineosin-stainedsection of follicular lymphoma shows mostly small centrocytes with cleaved nuclei and an occasional centroblast (arrow) with a large nucleus and peripheral nucleoli. Inset shows the closer, less well-defined arrangement of several follicles. (Middle left) CD20 stains B cells within the follicles of RLH. The inset indicates the substantial width of the interfollicular zones. (Middle right) CD20 also stains the B cells in the follicles of follicular lymphoma; the close arrangement of the follicles can again be appreciated in the inset.(Bottom left) CD3 stains T cells in the interfollicular zones (inset); the T cells can also be seen percolating throughout the folliclesof RLH. (Bottom right) Follicular lymphoma manifests nearly identical CD3 staining, with CD3\_ cells prominent in the interfollicular zones (inset) as well as scattered within the follicles. (Top left and top right, hematoxylineosin, 400, insets, 25;Middle left through Bottom right, immunoperoxidase reactions, diaminobenzidine chromogen, 200, insets 25) [42].

Stratification of Patients with Follicular Lymphoma 379

Fig. 3. Further immunohistochemical characterization of reactive lymphoid hyperplasia (RLH) and follicular lymphoma. (Topleft) CD20 densely stains this region of RLH without obvious follicular architecture. (Top right) CD23 highlights the dendritic cell scaffolding of

broughtout by CD23 in follicular lymphoma. (Bottom left) Ki-67 stains cells within follicles of RLH in an uneven distribution; staining is crescentic and more dense at the bottom of the follicle. (Bottom right) Follicular cells in follicular lymphoma are more evenly and diffusely positive for Ki-67.(Immunoperoxidase reactions, diaminobenzidine chromogen, 200, inset,

centrocytes and centroblasts. Evidence of the t(14;18) is uncommon and most cases are BCL2 negative. Dissemination beyond the skin is rare, and the prognosis is usually excellent [47]. However, most studies have shown poor interobserver and intraobserver reproducibility. Moreover, the clinical significance of the separation of Grades 1 and 2 has been questioned, with minimal differences seen in long term outcome. Thus, the 2008 WHO classification lumps cases with few centroblasts as "FL Grade 1-2 (low grade)" and does not require or recommend further separation. FL Grade 3 is divided into Grades 3A and 3B, based on the absence of centrocytes in the latter category. Several studies have identified biological differences between these two subtypes, with most cases of FL Grade 3B being more closely

the follicles in an adjacent section. The inset shows a similar dendritic architecture

25) [42].

related to DLBCL at the molecular level [47].

Fig. 2. Immunohistochemical characterization of reactive lymphoid hyperplasia and follicular lymphoma. (Top left) BCL-6stains B cells within follicles of reactive lymphoid hyperplasia (RLH). (Top right) Follicles of follicular lymphoma are also positivefor BCL-6. (Middle left) The follicles of RLH are negative for BCL-2. (Middle right) Follicles of follicular lymphoma are positivefor BCL-2. (Bottom left) Follicles in RLH are positive for CD10. (Bottom right) Follicles (F) of follicular lymphoma are also positive for CD10 but with more interfollicular staining than RLH. (Immunoperoxidase reactions, diaminobenzidine chromogen,200) [42].

#### **8. Follicular lymphoma—How many grades?**

The grading of FL was the subject of spirited discussion, both among the authors and the participants in the Clinical Advisory Committee. FL has traditionally been graded according to the proportion of centroblasts and into three Grades, 1-3 as detailed:

World Health Organization classification of follicular lymphoma (FL).

Follicular Lymphoma: Grading & Variants

Grade 1: 0–5 centroblasts/HPF Grade 2: 6–15 centroblasts/HPF Grade 3: > 15 centroblasts/HPF

3a: > 15 centroblasts, but centrocytes are still present 3b: centroblasts form solid sheets with no residual centrocytes [46].

Variants:

Primary cutaneous follicle center lymphoma Pediatric follicle center lymphoma Intestinal follicle center lymphoma Diffuse follicle center lymphoma Grade 1, 0–5 centroblasts/HPF Grade 2, 6–15centroblasts/HPF[43]

Pediatric FL lacking an association with the t(14;18). Primary cutaneous follicle center lymphoma (PCFCL) may contain a high proportion of large B-cells including large

Fig. 2. Immunohistochemical characterization of reactive lymphoid hyperplasia and follicular lymphoma. (Top left) BCL-6stains B cells within follicles of reactive lymphoid hyperplasia (RLH). (Top right) Follicles of follicular lymphoma are also positivefor BCL-6. (Middle left) The follicles of RLH are negative for BCL-2. (Middle right) Follicles of follicular lymphoma are positivefor BCL-2. (Bottom left) Follicles in RLH are positive for CD10. (Bottom right) Follicles (F) of follicular lymphoma are also positive for CD10 but with more

interfollicular staining than RLH. (Immunoperoxidase reactions, diaminobenzidine

to the proportion of centroblasts and into three Grades, 1-3 as detailed: World Health Organization classification of follicular lymphoma (FL).

3b: centroblasts form solid sheets with no residual centrocytes [46].

The grading of FL was the subject of spirited discussion, both among the authors and the participants in the Clinical Advisory Committee. FL has traditionally been graded according

Pediatric FL lacking an association with the t(14;18). Primary cutaneous follicle center lymphoma (PCFCL) may contain a high proportion of large B-cells including large

**8. Follicular lymphoma—How many grades?**

3a: > 15 centroblasts, but centrocytes are still present

Follicular Lymphoma: Grading & Variants

Primary cutaneous follicle center lymphoma

Pediatric follicle center lymphoma Intestinal follicle center lymphoma Diffuse follicle center lymphoma Grade 1, 0–5 centroblasts/HPF Grade 2, 6–15centroblasts/HPF[43]

Grade 1: 0–5 centroblasts/HPF Grade 2: 6–15 centroblasts/HPF Grade 3: > 15 centroblasts/HPF

Variants:

chromogen,200) [42].

Fig. 3. Further immunohistochemical characterization of reactive lymphoid hyperplasia (RLH) and follicular lymphoma. (Topleft) CD20 densely stains this region of RLH without obvious follicular architecture. (Top right) CD23 highlights the dendritic cell scaffolding of the follicles in an adjacent section. The inset shows a similar dendritic architecture broughtout by CD23 in follicular lymphoma. (Bottom left) Ki-67 stains cells within follicles of RLH in an uneven distribution; staining is crescentic and more dense at the bottom of the follicle. (Bottom right) Follicular cells in follicular lymphoma are more evenly and diffusely positive for Ki-67.(Immunoperoxidase reactions, diaminobenzidine chromogen, 200, inset, 25) [42].

centrocytes and centroblasts. Evidence of the t(14;18) is uncommon and most cases are BCL2 negative. Dissemination beyond the skin is rare, and the prognosis is usually excellent [47].

However, most studies have shown poor interobserver and intraobserver reproducibility. Moreover, the clinical significance of the separation of Grades 1 and 2 has been questioned, with minimal differences seen in long term outcome. Thus, the 2008 WHO classification lumps cases with few centroblasts as "FL Grade 1-2 (low grade)" and does not require or recommend further separation. FL Grade 3 is divided into Grades 3A and 3B, based on the absence of centrocytes in the latter category. Several studies have identified biological differences between these two subtypes, with most cases of FL Grade 3B being more closely related to DLBCL at the molecular level [47].

Stratification of Patients with Follicular Lymphoma 381

occur in some lymph nodes while low-grade lymphoma persists in others. An advantage of FNA is the ability to sample multiple lymph nodes, whereas open biopsy of different

Grading FL is based on the proportion of centroblasts in neoplastic follicles; therefore, the ability to identify the various cellular components in a fine-needle aspirate is the first essential step to the grading process. In Papanicolaou stained preparations, centrocytes or cleaved follicular center cells are small to medium-sized cells with angulated, elongated,

Centroblasts are at least two times larger than a lymphocyte and usually are round or oval but occasionally have indented, irregular, or even lobulated nuclei. There is a narrow rim of cytoplasm, often basophilic to amphophilic. A large and central nucleolus with chromatin clearing around it is characteristic of immunoblasts. In more typical centroblasts, the chromatin generally is vesicular with one to three prominent, often peripherally located nucleoli. The cells also may be hyperchromatic. Centroblasts tend to be more fragile and, in

It is important not to confuse centroblasts with follicular dendritic reticulum cells, which tend to aggregate within the center of the neoplastic follicles. Although dendritic cells have nuclei that are similar in size to centroblasts, the nuclei of dendritic cells are somewhat coffee bean-shaped with one side typically flattened and with fine, smooth nuclear membranes. The cytoplasm is indistinct, not basophilic, in contrast to that of centroblasts. In Papanicolaou-stained preparations, the chromatin is pale gray and finely granular with small, central, eosinophilic nucleoli. The cytoplasm of dendritic cells form long, dendritic processes that can be appreciated in cell blocks by IHC staining for CD21. Large cleaved cells also must be distinguished from centroblasts. Although there is an overlap in size with centroblasts, large centrocytes are more irregular in shape and lack the prominent nucleoli

When it comes to differentiating individual cells, cytologic preparations are superior to hematoxylin and eosin-stained histologic sections, although cytologic preparations typically

Cell blocks are complimentary to smears and provide additional architectural clues. The FNA process often aspirates intact follicular structures that can be appreciated in the cell block. The presence of intact follicles may be proven by special stains on sections of the cell

All three grades of lymphoma can have varying proportions of follicular and diffuse areas. Grade 1 and 2 FL generally have a predominantly follicular pattern. Because they are better

Grade 3 FL occurs less frequently than Grade 1 and 2 FL, and a pure follicular pattern in Grade 3 FL is even more unusual. In Grade 3 FL, the presence of diffuse areas is more common, and most (but not all) studies show that this finding is associated with a worse

differentiated, they have retained the ability to recapitulate follicles [ 51].

twisted, or cleaved nuclei with inconspicuous nucleoli and scant, pale cytoplasm.

anatomic sites is not feasible

some preparations, may not be preserved well [50].

and chromatin pattern of centroblasts [50]

are less informative about architecture.

**10. Grade versus pattern**

block [50].

prognosis.

However, in clinical practice the separation of Grades 3A and 3B can be challenging. Diffuse areas in any Grade 3 FL should be designated as DLBCL (with FL) and are more commonly observed in Grade 3B. Further studies are likely to lead to more precise delineation of the Grade 3 cases truly belonging within FL and those representing an intrafollicular variant of the GCB (germinal center B cell) type of DLBCL.

The presence of diffuse areas within a FL appears to confer more aggressive clinical behavior. In addition, unusual cytological variants can be encountered that are not included in the World Health Organization classification, including cases with large centrocytes and others with small centroblasts; in some cases the latter may resemble the cytomorphology of Burkitt lymphoma and may be associated with MYC oncogene translocations [43].

A truly diffuse form of FL may be rarely encountered, but on closer inspection, most cases demonstrate vaguely follicular architecture that is underappreciated without the routine use of immunostains for follicular dendritic cells. When a diagnosis of true diffuse follicle center lymphoma is considered, the pathologist is encouraged to demonstrate co-expression of CD10 and Bcl-6 as well as presumptive evidence of the t(14;18). Approximately 10% of FL cases reveal the presence of a zone of cells resembling marginal zone B cells, immediately surrounding neoplastic follicles. Importantly, a residual benign mantle zone is not seen, helping to distinguish FL from a marginal zone lymphoma. The area of marginal zone differentiation within a FL takes on a distinct morphology with cells having moderate amount of pale cytoplasm; moreover, the immunophenotype is also different, with downregulation of CD10 and Bcl-6 expression by cells within the marginal zone compartment [48].

Unresolved issues in FL pathology remain and to some extent contribute to problems with reproducibility. Diffuse areas in FL are often not identified, due to underutilization of follicular dendritic cell stains. Similarly, follicular areas in suspected de novo DLBCL are underappreciated. Variability in the cytology of cells within follicles contributes to inconsistent reporting of FL grade. Follicular dendritic are commonly misidentified as centroblasts, resulting in higher grade and, in general, there is poor interobserver reproducibility for the counting of centroblasts. All of these factors lead to inconsistencies in the diagnosis and grading of FL biopsies, and to some extent raise questions regarding the validity of grading and clinical decision making based on grade [43].

Finally, a rare form of FL may be seen referred to as "in-situ" FL. In these uncommon cases, scattered malignant follicles are identified within lymph nodes revealing mostly benign features. The malignant follicles show typically more monomorphic germinal centres (GCs). GCs involved in intrafollicular neoplasia/in situ follicular lymphoma are composed mainly of centrocytes with no evident atypia. A feature of these centres is the relative absence of macrophages with tingible bodies and the absence of polarization into light and dark zones. The paucity of large centroblasts reflects their low proliferation rate in insitu FLs. Intrafollicular neoplasia/in situ follicular lymphoma is characterized by strong coexpression of Bcl-2 and CD10 in the involved GCs [49].

### **9. Fine needle aspiration cytology**

FNA is a useful tool for staging as well as evaluating recurrences in lymph nodes and extra nodal sites without subjecting patients to multiple excisional biopsies. Transformation can

However, in clinical practice the separation of Grades 3A and 3B can be challenging. Diffuse areas in any Grade 3 FL should be designated as DLBCL (with FL) and are more commonly observed in Grade 3B. Further studies are likely to lead to more precise delineation of the Grade 3 cases truly belonging within FL and those representing an intrafollicular variant of

The presence of diffuse areas within a FL appears to confer more aggressive clinical behavior. In addition, unusual cytological variants can be encountered that are not included in the World Health Organization classification, including cases with large centrocytes and others with small centroblasts; in some cases the latter may resemble the cytomorphology of

A truly diffuse form of FL may be rarely encountered, but on closer inspection, most cases demonstrate vaguely follicular architecture that is underappreciated without the routine use of immunostains for follicular dendritic cells. When a diagnosis of true diffuse follicle center lymphoma is considered, the pathologist is encouraged to demonstrate co-expression of CD10 and Bcl-6 as well as presumptive evidence of the t(14;18). Approximately 10% of FL cases reveal the presence of a zone of cells resembling marginal zone B cells, immediately surrounding neoplastic follicles. Importantly, a residual benign mantle zone is not seen, helping to distinguish FL from a marginal zone lymphoma. The area of marginal zone differentiation within a FL takes on a distinct morphology with cells having moderate amount of pale cytoplasm; moreover, the immunophenotype is also different, with downregulation of CD10 and Bcl-6 expression by cells within the marginal zone

Unresolved issues in FL pathology remain and to some extent contribute to problems with reproducibility. Diffuse areas in FL are often not identified, due to underutilization of follicular dendritic cell stains. Similarly, follicular areas in suspected de novo DLBCL are underappreciated. Variability in the cytology of cells within follicles contributes to inconsistent reporting of FL grade. Follicular dendritic are commonly misidentified as centroblasts, resulting in higher grade and, in general, there is poor interobserver reproducibility for the counting of centroblasts. All of these factors lead to inconsistencies in the diagnosis and grading of FL biopsies, and to some extent raise questions regarding the

Finally, a rare form of FL may be seen referred to as "in-situ" FL. In these uncommon cases, scattered malignant follicles are identified within lymph nodes revealing mostly benign features. The malignant follicles show typically more monomorphic germinal centres (GCs). GCs involved in intrafollicular neoplasia/in situ follicular lymphoma are composed mainly of centrocytes with no evident atypia. A feature of these centres is the relative absence of macrophages with tingible bodies and the absence of polarization into light and dark zones. The paucity of large centroblasts reflects their low proliferation rate in insitu FLs. Intrafollicular neoplasia/in situ follicular lymphoma is characterized by strong co-

FNA is a useful tool for staging as well as evaluating recurrences in lymph nodes and extra nodal sites without subjecting patients to multiple excisional biopsies. Transformation can

validity of grading and clinical decision making based on grade [43].

expression of Bcl-2 and CD10 in the involved GCs [49].

**9. Fine needle aspiration cytology**

Burkitt lymphoma and may be associated with MYC oncogene translocations [43].

the GCB (germinal center B cell) type of DLBCL.

compartment [48].

occur in some lymph nodes while low-grade lymphoma persists in others. An advantage of FNA is the ability to sample multiple lymph nodes, whereas open biopsy of different anatomic sites is not feasible

Grading FL is based on the proportion of centroblasts in neoplastic follicles; therefore, the ability to identify the various cellular components in a fine-needle aspirate is the first essential step to the grading process. In Papanicolaou stained preparations, centrocytes or cleaved follicular center cells are small to medium-sized cells with angulated, elongated, twisted, or cleaved nuclei with inconspicuous nucleoli and scant, pale cytoplasm.

Centroblasts are at least two times larger than a lymphocyte and usually are round or oval but occasionally have indented, irregular, or even lobulated nuclei. There is a narrow rim of cytoplasm, often basophilic to amphophilic. A large and central nucleolus with chromatin clearing around it is characteristic of immunoblasts. In more typical centroblasts, the chromatin generally is vesicular with one to three prominent, often peripherally located nucleoli. The cells also may be hyperchromatic. Centroblasts tend to be more fragile and, in some preparations, may not be preserved well [50].

It is important not to confuse centroblasts with follicular dendritic reticulum cells, which tend to aggregate within the center of the neoplastic follicles. Although dendritic cells have nuclei that are similar in size to centroblasts, the nuclei of dendritic cells are somewhat coffee bean-shaped with one side typically flattened and with fine, smooth nuclear membranes. The cytoplasm is indistinct, not basophilic, in contrast to that of centroblasts. In Papanicolaou-stained preparations, the chromatin is pale gray and finely granular with small, central, eosinophilic nucleoli. The cytoplasm of dendritic cells form long, dendritic processes that can be appreciated in cell blocks by IHC staining for CD21. Large cleaved cells also must be distinguished from centroblasts. Although there is an overlap in size with centroblasts, large centrocytes are more irregular in shape and lack the prominent nucleoli and chromatin pattern of centroblasts [50]

When it comes to differentiating individual cells, cytologic preparations are superior to hematoxylin and eosin-stained histologic sections, although cytologic preparations typically are less informative about architecture.

Cell blocks are complimentary to smears and provide additional architectural clues. The FNA process often aspirates intact follicular structures that can be appreciated in the cell block. The presence of intact follicles may be proven by special stains on sections of the cell block [50].

### **10. Grade versus pattern**

All three grades of lymphoma can have varying proportions of follicular and diffuse areas. Grade 1 and 2 FL generally have a predominantly follicular pattern. Because they are better differentiated, they have retained the ability to recapitulate follicles [ 51].

Grade 3 FL occurs less frequently than Grade 1 and 2 FL, and a pure follicular pattern in Grade 3 FL is even more unusual. In Grade 3 FL, the presence of diffuse areas is more common, and most (but not all) studies show that this finding is associated with a worse prognosis.

Stratification of Patients with Follicular Lymphoma 383

instability. This may, in turn, foster the occurrence of secondary oncogenic hits and, finally,

Cong and co-workers[58],described the phenomenon of what they termed follicular lymphoma in situ in otherwise reactive, hyperplastic lymph nodes possibly representing the

A number of secondary chromosomal alterations have been described in FL including: structural and numerical changes. The complexity of the secondary alterations correlates with the grade – the higher the grade, the more complex aberrations are usually

It has long been recognized that these alterations occur in a non-random fashion. Partial trisomies of chromosomes 1q, 7, 8 and 18q, and deletions in 1p and 6q have been described as the most common secondary alterations, and deletions in the long arm of chromosomes 1 and 6 and in the short arm of chromosome 17 have been associated with a worse prognosis [60]. Some of these alterations may occur early in the course of the disease, whereas others might represent late genetic events. In addition, some of the alterations are mutually exclusive, while alterations of other chromosomal regions frequently appear together

Some of the secondary chromosomal alterations may cancel the effect of the t(14;18) that initially forms a low-grade neoplasia with a follicular growth pattern and subsequently enable the transformation to highgrade lymphoma. This process has been associated with three distinct secondary genetic alterations in FL that have a profound impact on the biological program and the clinical course in FL. These include an additional introduction of a t(8;14)/MYC rearrangement in the tumor cells [62], the inactivation of TP53 by mutation and deletion and, finally, the inactivation of p16, frequently occurring by biallelic deletion

The occurrence of a secondary MYC rearrangement in FL deserves particular attention, because these cases frequently demonstrate a Burkitt-like appearance and may be detectable by virtue of this specific morphology in combination with an overexpression of the BCL2 protein caused by the t(14;18) that is usually not encountered in classical Burkitt's lymphomas. Some studies suggest that the detection of TP53 mutations in primary diagnostic specimens of FL without signs of transformation also characterizes a patient

From recent studies t(14;18)-negative FLs belong to the biologic spectrum of FL, but show distinct genetic features as well as gene expression and immunohistochemical profiles that differ from their t(14;18)-positive counterparts [65]. The t(14;18)-negative FL appears to harbor genetic rearrangements of the BCL6 gene in 3q27[66] or trisomy 3 [67] whereas others show BCL2 expression on the immunohistochemical level despite the lack of the t(14;18)[68]. Moreover, increased expression of IRF4/MUM1, a protein associated with plasma cell differentiation has been described in FL without BCL2 rearrangement [69].

**12. Secondary chromosomal aberrations in follicular lymphoma**

possibly leading to a coordinated deregulation of genetic pathways [61].

result in the malignant transformation to overt FL [57].

morphological equivalent of early, pre-invasive FL.

encountered [59].

[63].

subgroup with worse prognosis [64].

**13. BCL2-negative follicular lymphoma**

In Grade 1 and 2 FL, there is conflicting evidence regarding whether the presence of large, diffuse areas or the degree of nodularity may alter prognosis significantly.

SO the WHO classification system recommends estimating the proportion of follicular and diffuse components in the pathology report

Note, however, that the proportion of centroblasts within the neoplastic follicles is what determines the grade of a FL, not the degree of nodularity. Furthermore, the grade of FL, in combination with other clinical factors,ultimately is what influences treatment decisions [50].

#### **11. Cytogenetics of follicular lymphoma**

The t(14;18)(q32;q21) chromosome translocation represents the defining cytogenetic hallmark of FL and is encountered in 80%-90% of cases. Its molecular consequence is the juxtaposition of the B-cell lymphoma/leukemia 2 (BCL2) proto-oncogene with enhancer sequences of the immunoglobulin heavy chain gene (IGH) promoter region, thereby deregulating its expression and resulting in an overexpression of the BCL2 protein in the neoplastic follicles [52,53]. However, 10–15% of cases do not harbor the t(14;18)(q32;q21) and in these t(14;18)-negative cases, other mechanisms are thought to be involved in the pathogenesis [54]. Moreover, t(14;18)- positive B cells can be identified in the blood and lymphoid tissues of healthy individuals, and the number of t(14;18)-positive cells is influenced by gender, personal lifestyle and exposure to toxic substances [55].

The BCL2 proto oncogene, a potent anti-apoptotic molecule, is expressed in resting B cells in the perifollicular mantle zone and in post-follicular B cells, thereby promoting long-lived follicular precursor and memory B cells. Germinal center B cells, however, physiologically lack BCL2 expression and undergo apoptosis unless they are selected by specific antigens that drive them into processes termed somatic hypermutation and class switching. Due to the lack of BCL2 expression, amongst other factors, the large bulk of B-cells entering the GC microenvironment will be removed by apoptosis. The constitutive overexpression of BCL2 in germinal center B cells inferred by the t(14;18)(q32;q21) leads to an accumulation of inappropriately rescued B cells with a prolonged life span, allowing for the development of additional genetic hits to occur, that are required for the establishment of overt FL . Variant translocations of the t(14;18), such as the t(2;18) or t(18;22), juxtapose BCL2 to the loci of the immunoglobulin light chains (k,l) and, likewise, result in inappropriate and sustained BCL2 expression in GC B cells [56].

The occurrence of the t(14;18) in a pre-FL B cell can be viewed as a first hit in a multistep process that results in the clonal dysregulation of cell cycle control and apoptosis of the tumor cells. During process of lymphomagenesis, a number of additional genetic or epigenetic events occur in a non-random fashion that lead to overt FL .For example, constitutive expression of activation-induced cytidine deaminase (AID) in the GC environment in B cells overexpressing BCL2 may propagate continuous somatic hypermutation and class switch recombination activity that results in increased genomic

In Grade 1 and 2 FL, there is conflicting evidence regarding whether the presence of large,

SO the WHO classification system recommends estimating the proportion of follicular and

Note, however, that the proportion of centroblasts within the neoplastic follicles is what determines the grade of a FL, not the degree of nodularity. Furthermore, the grade of FL, in combination with other clinical factors,ultimately is what influences treatment decisions

The t(14;18)(q32;q21) chromosome translocation represents the defining cytogenetic hallmark of FL and is encountered in 80%-90% of cases. Its molecular consequence is the juxtaposition of the B-cell lymphoma/leukemia 2 (BCL2) proto-oncogene with enhancer sequences of the immunoglobulin heavy chain gene (IGH) promoter region, thereby deregulating its expression and resulting in an overexpression of the BCL2 protein in the neoplastic follicles [52,53]. However, 10–15% of cases do not harbor the t(14;18)(q32;q21) and in these t(14;18)-negative cases, other mechanisms are thought to be involved in the pathogenesis [54]. Moreover, t(14;18)- positive B cells can be identified in the blood and lymphoid tissues of healthy individuals, and the number of t(14;18)-positive cells is

The BCL2 proto oncogene, a potent anti-apoptotic molecule, is expressed in resting B cells in the perifollicular mantle zone and in post-follicular B cells, thereby promoting long-lived follicular precursor and memory B cells. Germinal center B cells, however, physiologically lack BCL2 expression and undergo apoptosis unless they are selected by specific antigens that drive them into processes termed somatic hypermutation and class switching. Due to the lack of BCL2 expression, amongst other factors, the large bulk of B-cells entering the GC microenvironment will be removed by apoptosis. The constitutive overexpression of BCL2 in germinal center B cells inferred by the t(14;18)(q32;q21) leads to an accumulation of inappropriately rescued B cells with a prolonged life span, allowing for the development of additional genetic hits to occur, that are required for the establishment of overt FL . Variant translocations of the t(14;18), such as the t(2;18) or t(18;22), juxtapose BCL2 to the loci of the immunoglobulin light chains (k,l) and, likewise, result in inappropriate and sustained BCL2

The occurrence of the t(14;18) in a pre-FL B cell can be viewed as a first hit in a multistep process that results in the clonal dysregulation of cell cycle control and apoptosis of the tumor cells. During process of lymphomagenesis, a number of additional genetic or epigenetic events occur in a non-random fashion that lead to overt FL .For example, constitutive expression of activation-induced cytidine deaminase (AID) in the GC environment in B cells overexpressing BCL2 may propagate continuous somatic hypermutation and class switch recombination activity that results in increased genomic

influenced by gender, personal lifestyle and exposure to toxic substances [55].

diffuse areas or the degree of nodularity may alter prognosis significantly.

diffuse components in the pathology report

**11. Cytogenetics of follicular lymphoma**

expression in GC B cells [56].

[50].

instability. This may, in turn, foster the occurrence of secondary oncogenic hits and, finally, result in the malignant transformation to overt FL [57].

Cong and co-workers[58],described the phenomenon of what they termed follicular lymphoma in situ in otherwise reactive, hyperplastic lymph nodes possibly representing the morphological equivalent of early, pre-invasive FL.

### **12. Secondary chromosomal aberrations in follicular lymphoma**

A number of secondary chromosomal alterations have been described in FL including: structural and numerical changes. The complexity of the secondary alterations correlates with the grade – the higher the grade, the more complex aberrations are usually encountered [59].

It has long been recognized that these alterations occur in a non-random fashion. Partial trisomies of chromosomes 1q, 7, 8 and 18q, and deletions in 1p and 6q have been described as the most common secondary alterations, and deletions in the long arm of chromosomes 1 and 6 and in the short arm of chromosome 17 have been associated with a worse prognosis [60]. Some of these alterations may occur early in the course of the disease, whereas others might represent late genetic events. In addition, some of the alterations are mutually exclusive, while alterations of other chromosomal regions frequently appear together possibly leading to a coordinated deregulation of genetic pathways [61].

Some of the secondary chromosomal alterations may cancel the effect of the t(14;18) that initially forms a low-grade neoplasia with a follicular growth pattern and subsequently enable the transformation to highgrade lymphoma. This process has been associated with three distinct secondary genetic alterations in FL that have a profound impact on the biological program and the clinical course in FL. These include an additional introduction of a t(8;14)/MYC rearrangement in the tumor cells [62], the inactivation of TP53 by mutation and deletion and, finally, the inactivation of p16, frequently occurring by biallelic deletion [63].

The occurrence of a secondary MYC rearrangement in FL deserves particular attention, because these cases frequently demonstrate a Burkitt-like appearance and may be detectable by virtue of this specific morphology in combination with an overexpression of the BCL2 protein caused by the t(14;18) that is usually not encountered in classical Burkitt's lymphomas. Some studies suggest that the detection of TP53 mutations in primary diagnostic specimens of FL without signs of transformation also characterizes a patient subgroup with worse prognosis [64].

### **13. BCL2-negative follicular lymphoma**

From recent studies t(14;18)-negative FLs belong to the biologic spectrum of FL, but show distinct genetic features as well as gene expression and immunohistochemical profiles that differ from their t(14;18)-positive counterparts [65]. The t(14;18)-negative FL appears to harbor genetic rearrangements of the BCL6 gene in 3q27[66] or trisomy 3 [67] whereas others show BCL2 expression on the immunohistochemical level despite the lack of the t(14;18)[68]. Moreover, increased expression of IRF4/MUM1, a protein associated with plasma cell differentiation has been described in FL without BCL2 rearrangement [69].

Stratification of Patients with Follicular Lymphoma 385

compared to gold standard approaches, PCR-based techniques only detect up to 60% of translocations, which seriously impairs the diagnostic capability of PCR. However, BIOMED-2 primers have been developed using three multiplex tubes for detection of MBR-JH, 3'MBR- JH, and mcr-JH to maximize the detection of t(14;18) [73]. ). These data are supported by previous report from our molecular hematology laboratory. We found that FISH was superior to PCR in the detection of t(14'18) (q32'q21)-IGH-BCL-2 in formalin-fixed, paraffin-embedded tissue samples. Moreover, strong correlations between the FLIPI score

Molecular profiling of many types of lymphoma using RQ-PCR and cDNA microarray has been used to predict survival by many researchers [91-94]. Genes involved in cell cycle control and DNA synthesis and metabolism (e.g. CXCL12, which is involved in signaling transduction and NEK2, which is involved in mitotic regulation, and MAPK1) are significantly up-regulated in the aggressive phase of FL[3]. MYC, as a known oncogene, and MYC-target genes (SFRS7, LDHA, MTHFD1, NME1, MSH2, and CKS2) are upregulated on transformation and may be implicated as a direct transforming factor [95-102].On the other hand, there is higher density of the T-cell infiltrate in low-grade FL as compared to highgrade disease and this is reflected by several T cell–related genes (CD3, CD2, CD69). However, genes related to T-cell and macrophage activation including several chemokine receptors (CCR1, CCL3, CCL5, CCL8, AKAP12, ILF3, GEM) are significantly upregulated on transformation, suggesting an important biologic role. Notably, specific antagonists to several of the above-mentioned chemokine receptors are available and offer an attractive

The National Comprehensive Cancer Network( NCCN) has recently launched an algorithm

and each of interphase FISH and CD10 expression were demonstrated [90].

**15. Proposed algorithm for stratification of follicular lymphoma**

Fig. 4. Proposed algorithm for stratification of follicular lymphoma (NCCN)

possibility for therapeutic interventions [103].

for stratification of follicular lymphoma (Figure 4).

### **14. Molecular genetics of follicular lymphoma**

Immunoglobulin heavy and light chains are rearranged in FL with the variable region genes showing extensive and ongoing somatic hypermutation [70, 71]. As a result of these mutations in the CDR-regions, PCR primer annealing may be hampered and depending on the primers used, immunoglobulin-PCR may not yield monoclonal products in a proportion of FL cases (10-40%). Multiplex PCR reactions using BIOMED-2 expanded primer sets detect closer to 90% of complete *IGH* (VH-JH) gene rearrangements, and clonality detection approximates 100% when primers detecting incomplete *IGH* (DH-JH) and light chain gene rearrangements are included [72].

For amplification of complete *IGH* (VH-JH) gene rearrangement, BIOMED-2 developed three sets of VH primers corresponding to the three VH FR regions (FR1, FR2, and FR3). Each set of primers consisted of six or seven oligonucleotides capable of annealing to their corresponding VH segments (VH1–VH7) with no mismatches for most VH segments. These VH primer sets were used in conjunction with a single JH consensus primer. The JH primer is fluorescently labeled to allow the detection of PCR products by Gene Scanning [73].

For incomplete *IGH* (DH-JH) rearrangements, seven family-specific DH primers were designed based on the high degree of homology within each DH family in combination with the consensus JH primer. Primers were designed such that crossannealing to other DH family segments would be minimal or preferably absent [73].

Six family-specific Vk primers were designed by van Dongen et al [73], to recognize the various Vk gene segments of the seven Vk families. The family-specific Vk primers were designed to be used in combination with either a set of two Jk primers or a Kde primer.A single consensus primer recognizing both Vλ1 and Vλ2 gene segments, as well as a Vλ3 primer, were designed by van Dongen et al [73], in combination to a single consensus primer for the Jλ1, Jλ2, and Jλ3 gene segments.

The *t(14;18)* and *BCL2* gene rearrangements is one of the best characterized recurrent cytogenetic abnormalities in peripheral B-cell lymphoproliferative disease[74] . FL is genetically characterized by this translocation which is present in up to 90% of the grade 1-2 FL cases [75,76] but the proportion depends on the technique used [77, 78, 79]. BCL2 rearrangements are much less frequent in grade 3B FL [80]. As a consequence of the translocation, the *BCL2* gene (anti-apoptotic) from 18q21 is placed under the control of the strong enhancers of the *IGH* locus resulting in the deregulation of its normal pattern of expression [81,82]. The *BCL2/IGH* rearrangement is found in the PB of 25-75% of healthy donors, and also in reactive nodes, particularly if using sensitive nested or RT-PCR assays [83,84,85]. A recent study suggests that rather than being naive B-cells, these *BCL2*-rearranged cells are memory B-cells [86].

There is no single gold standard detection strategy for the *t(14;18)*, and a combination of cytogenetics and southern blotting have been generally used [87,88]. Interphase FISH detection strategies offer an applicable alternative that have the potential to pick up more translocations [89]. For molecular diagnostic laboratories PCR-based detection strategies offer rapid results, are generally applicable, and can be used for residual disease monitoring. However, the primers commonly used have not been designed to take into account recent information on the molecular anatomy of the breakpoints. As a consequence when

Immunoglobulin heavy and light chains are rearranged in FL with the variable region genes showing extensive and ongoing somatic hypermutation [70, 71]. As a result of these mutations in the CDR-regions, PCR primer annealing may be hampered and depending on the primers used, immunoglobulin-PCR may not yield monoclonal products in a proportion of FL cases (10-40%). Multiplex PCR reactions using BIOMED-2 expanded primer sets detect closer to 90% of complete *IGH* (VH-JH) gene rearrangements, and clonality detection approximates 100% when primers detecting incomplete *IGH* (DH-JH) and light chain gene

For amplification of complete *IGH* (VH-JH) gene rearrangement, BIOMED-2 developed three sets of VH primers corresponding to the three VH FR regions (FR1, FR2, and FR3). Each set of primers consisted of six or seven oligonucleotides capable of annealing to their corresponding VH segments (VH1–VH7) with no mismatches for most VH segments. These VH primer sets were used in conjunction with a single JH consensus primer. The JH primer is fluorescently labeled to allow the detection of PCR products by Gene Scanning [73].

For incomplete *IGH* (DH-JH) rearrangements, seven family-specific DH primers were designed based on the high degree of homology within each DH family in combination with the consensus JH primer. Primers were designed such that crossannealing to other DH

Six family-specific Vk primers were designed by van Dongen et al [73], to recognize the various Vk gene segments of the seven Vk families. The family-specific Vk primers were designed to be used in combination with either a set of two Jk primers or a Kde primer.A single consensus primer recognizing both Vλ1 and Vλ2 gene segments, as well as a Vλ3 primer, were designed by van Dongen et al [73], in combination to a single consensus

The *t(14;18)* and *BCL2* gene rearrangements is one of the best characterized recurrent cytogenetic abnormalities in peripheral B-cell lymphoproliferative disease[74] . FL is genetically characterized by this translocation which is present in up to 90% of the grade 1-2 FL cases [75,76] but the proportion depends on the technique used [77, 78, 79]. BCL2 rearrangements are much less frequent in grade 3B FL [80]. As a consequence of the translocation, the *BCL2* gene (anti-apoptotic) from 18q21 is placed under the control of the strong enhancers of the *IGH* locus resulting in the deregulation of its normal pattern of expression [81,82]. The *BCL2/IGH* rearrangement is found in the PB of 25-75% of healthy donors, and also in reactive nodes, particularly if using sensitive nested or RT-PCR assays [83,84,85]. A recent study suggests that rather than being naive B-cells, these *BCL2*-re-

There is no single gold standard detection strategy for the *t(14;18)*, and a combination of cytogenetics and southern blotting have been generally used [87,88]. Interphase FISH detection strategies offer an applicable alternative that have the potential to pick up more translocations [89]. For molecular diagnostic laboratories PCR-based detection strategies offer rapid results, are generally applicable, and can be used for residual disease monitoring. However, the primers commonly used have not been designed to take into account recent information on the molecular anatomy of the breakpoints. As a consequence when

**14. Molecular genetics of follicular lymphoma**

family segments would be minimal or preferably absent [73].

primer for the Jλ1, Jλ2, and Jλ3 gene segments.

arranged cells are memory B-cells [86].

rearrangements are included [72].

compared to gold standard approaches, PCR-based techniques only detect up to 60% of translocations, which seriously impairs the diagnostic capability of PCR. However, BIOMED-2 primers have been developed using three multiplex tubes for detection of MBR-JH, 3'MBR- JH, and mcr-JH to maximize the detection of t(14;18) [73]. ). These data are supported by previous report from our molecular hematology laboratory. We found that FISH was superior to PCR in the detection of t(14'18) (q32'q21)-IGH-BCL-2 in formalin-fixed, paraffin-embedded tissue samples. Moreover, strong correlations between the FLIPI score and each of interphase FISH and CD10 expression were demonstrated [90].

Molecular profiling of many types of lymphoma using RQ-PCR and cDNA microarray has been used to predict survival by many researchers [91-94]. Genes involved in cell cycle control and DNA synthesis and metabolism (e.g. CXCL12, which is involved in signaling transduction and NEK2, which is involved in mitotic regulation, and MAPK1) are significantly up-regulated in the aggressive phase of FL[3]. MYC, as a known oncogene, and MYC-target genes (SFRS7, LDHA, MTHFD1, NME1, MSH2, and CKS2) are upregulated on transformation and may be implicated as a direct transforming factor [95-102].On the other hand, there is higher density of the T-cell infiltrate in low-grade FL as compared to highgrade disease and this is reflected by several T cell–related genes (CD3, CD2, CD69). However, genes related to T-cell and macrophage activation including several chemokine receptors (CCR1, CCL3, CCL5, CCL8, AKAP12, ILF3, GEM) are significantly upregulated on transformation, suggesting an important biologic role. Notably, specific antagonists to several of the above-mentioned chemokine receptors are available and offer an attractive possibility for therapeutic interventions [103].

### **15. Proposed algorithm for stratification of follicular lymphoma**

The National Comprehensive Cancer Network( NCCN) has recently launched an algorithm for stratification of follicular lymphoma (Figure 4).

Fig. 4. Proposed algorithm for stratification of follicular lymphoma (NCCN)

Stratification of Patients with Follicular Lymphoma 387

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**15**

*1,2Spain 3UK*

Charles H. Lawrie

*1Biodonostia Institute, San Sebastián*

*2IKERBASQUE, Basque Foundation for Science, Bilbao*

**MicroRNA Expression in Follicular Lymphoma**

Lymphoma is the fifth most common cancer type in the Western world, accounting for approximately 12,000 cases per annum in the UK alone. Moreover the occurrence of this type of cancer has been increasing. The age-adjusted incidence of non-Hodgkin's lymphoma (NHL) in the US for example has increased 74% between 1976 and 2001 (SEER 2005). Follicular lymphoma (FL) is the most common form of low grade B-cell lymphoma (75-80% of all cases) representing about a third of all NHL cases in the US, and a quarter of all cases in Europe (Anderson et al. 1998). FL is characterised by the presence of the t(14;18) translocation in 90% of patients associated with up-regulation of the anti-apoptotic protein BCL2. Whilst FL tumours are chemo-sensitive, the disease is essentially incurable, with patients following a relapsing-remitting clinical course, typically experiencing several

Although indolent, with a median overall survival (OS) of ~10 years, about 30% of FL patients undergo high-grade transformation to an aggressive lymphoma that is histologically indistinguishable from diffuse large B-cell lympohoma (DLBCL). Transformed FL (tFL) patients have a particularly poor outcome with a median survival of <14 months (Wrench et al. 2010). The molecular basis of FL transformation is only poorly understood and importantly to date there are no reliable biomarkers that can identify FL patients at risk of transformation. In this chapter we will review the experimental evidence for the involvement of microRNAs in the pathology of FL with particular focus on the

FL is a neoplasm of follicle center B cells (centrocytes) characterized by a (partially) follicular growth pattern. FL tumor cells are believed to arise from normal germinal centre–associated (GC) B cells as they express the same antigen profile as GC B cells (i.e. CD19, CD20, CD10, BCL6, and membrane-bound IgM or IgG), share many morphological features of normal GC cells, and are found within a follicular architecture embedded in a network of T cells and follicular dendritic cells. In contrast to normal GC cells however, FL tumour cells are characterised by the presence of t(14:18) translocation resulting in expression of the anti-

episodes of disease before eventually becoming refractory to treatment.

**1. Introduction**

transformation process.

**2. Follicular lymphoma**

*3Nuffield Department of Clinical Laboratory Sciences, University of Oxford,*

profiling of follicular lymphoma and normal germinal center B cells using cDNA arrays. Blood, 99: 282-289

## **MicroRNA Expression in Follicular Lymphoma**

### Charles H. Lawrie

*1Biodonostia Institute, San Sebastián 2IKERBASQUE, Basque Foundation for Science, Bilbao 3Nuffield Department of Clinical Laboratory Sciences, University of Oxford, 1,2Spain 3UK*

### **1. Introduction**

392 Hematology – Science and Practice

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Muller-Hermelink HK, Smeland EB, Giltnane JM, Hurt EM, Zhao H, Averett L, Yang L, Wilson WH, Jaffe ES, Simon R, Klausner RD, Powell J, Duffey PL, Longo DL, Greiner TC, Weisenburger DD, Sanger WG, Dave BJ, Lynch JC, Vose J, Armitage JO, Montserrat E, López-Guillermo A, Grogan TM, Miller TP, LeBlanc M, Ott G, Kvaloy S, Delabie J, Holte H, Krajci P, Stokke T, Staudt LM (2002):. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-

Angelo M, Reich M, Pinkus GS, Ray TS, Koval MA, Last KW, Norton A, Lister TA, Mesirov J, Neuberg DS, Lander ES, Aster JC, Golub TR (2002): Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised

LFA, Joosten P, Kerkhoven RM, Bernards R, van Krieken JHJM, Kluin PM, van't Veer LJ and de Jong D (2005): Gene expression profiling in follicular lymphoma to assess clinical aggressiveness and to guide the choice of treatment. Blood, 105: 301-

lymphoma and normal germinal center B cells using cDNA arrays. Blood.;99:282-

signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy. Proc Natl Acad Sci U S A.;100:7259-7264. [99] Lossos IS, Alizadeh AA, Diehn M, et al (2002): Transformation of follicular lymphoma

to diffuse large-cell lymphoma: alternative patterns with increased or decreased expression of c-myc and its regulated genes. Proc Natl Acad Sci U S A.;99:8886-

Burkitt's, diffuse large-cell lymphoma of B-cell type associated with the c-

transcriptome by SAGE: identifcation and analysis of c-MYC target genes. Proc

Lymphoma is the fifth most common cancer type in the Western world, accounting for approximately 12,000 cases per annum in the UK alone. Moreover the occurrence of this type of cancer has been increasing. The age-adjusted incidence of non-Hodgkin's lymphoma (NHL) in the US for example has increased 74% between 1976 and 2001 (SEER 2005). Follicular lymphoma (FL) is the most common form of low grade B-cell lymphoma (75-80% of all cases) representing about a third of all NHL cases in the US, and a quarter of all cases in Europe (Anderson et al. 1998). FL is characterised by the presence of the t(14;18) translocation in 90% of patients associated with up-regulation of the anti-apoptotic protein BCL2. Whilst FL tumours are chemo-sensitive, the disease is essentially incurable, with patients following a relapsing-remitting clinical course, typically experiencing several episodes of disease before eventually becoming refractory to treatment.

Although indolent, with a median overall survival (OS) of ~10 years, about 30% of FL patients undergo high-grade transformation to an aggressive lymphoma that is histologically indistinguishable from diffuse large B-cell lympohoma (DLBCL). Transformed FL (tFL) patients have a particularly poor outcome with a median survival of <14 months (Wrench et al. 2010). The molecular basis of FL transformation is only poorly understood and importantly to date there are no reliable biomarkers that can identify FL patients at risk of transformation. In this chapter we will review the experimental evidence for the involvement of microRNAs in the pathology of FL with particular focus on the transformation process.

### **2. Follicular lymphoma**

FL is a neoplasm of follicle center B cells (centrocytes) characterized by a (partially) follicular growth pattern. FL tumor cells are believed to arise from normal germinal centre–associated (GC) B cells as they express the same antigen profile as GC B cells (i.e. CD19, CD20, CD10, BCL6, and membrane-bound IgM or IgG), share many morphological features of normal GC cells, and are found within a follicular architecture embedded in a network of T cells and follicular dendritic cells. In contrast to normal GC cells however, FL tumour cells are characterised by the presence of t(14:18) translocation resulting in expression of the anti-

MicroRNA Expression in Follicular Lymphoma 395

proliferation genes and followed an as yet undetermined route. In contrast a gene expression study of non-paired patients (24 FL patients who underwent transformation, 22 FL patients without transformation (after 7 years) and 24 DLBCL patients who had previously transformed from FL) found that gene expression was too heterogeneous to reliably predict transformation (Glas et al. 2007). They did however report a correlation by immunohistochemistry with the spatial distribution to neoplastic follicles and the activation of CD4+ T cells and specifically T-helper 1 cells (*P*>0.05). They did not find any correlation with other infiltrating cell populations including CD68+ macrophages or regulatory T cells. Additionally, genomic alterations have been demonstrated to be associated with transformation of FL. The acquisition of novel mutations in PIM-1, PAX-5, RhoH/TTF and c-MYC genes, due to aberrant somatic hypermutation, was found in 5/9 cases that had undergone transformation (Rossi et al. 2006). Genomic aberrations were found to be more common in transformed cases of DLBCL than non-transformed FL and the alterations -6q16-21 and +7pter-q22 were only found in transformed DLBCL but not in follicular lymphoma whereas -4q13-21 was more common in

However, despite intensive research the molecular basis for transformation in FL patients remains largely unknown. Recently, ourselves and others, have raised the possibility that microRNAs may be important factors in both FL transformation and antecedent FL

MicroRNAs are a recently discovered class of naturally occurring short non-coding RNA molecules that regulate eukaryotic gene expression post-transcriptionally. There are now more than 900 human microRNAs that have been identified through cloning and/or sequence analysis (miRBase- (Griffiths-Jones et al. 2006)), and it is believed some 60% of all human genes are a target for microRNA regulation (Friedman et al. 2009). MicroRNAs have been shown to play key regulatory roles in virtually every aspect of biology including developmental timing, cell differentiation, apoptosis, cell proliferation, metabolism organ development, and hematopoiesis (Kim 2005). The potential importance of microRNAs in cancer is implied by the finding that the majority of human microRNAs are located at cancer-associated genomic regions (Calin et al. 2004), and there is now overwhelming evidence that dysfunctional expression of microRNAs is a common, if not ubiquitous, feature of cancer in general and

Despite the fundamental role that microRNAs appear to play in biology, these molecules were unknown to the scientific world until 1993 when *lin-4*, a *C. elegans* developmental regulator was identified (Lee et al. 1993; Wightman et al. 1993). The significance of this finding was not however realised until seven years later when another worm microRNA, *let-7* was discovered (Reinhart et al. 2000). Unlike *lin-4*, the sequence of *let-7* was found to be highly conserved in almost all organisms (Pasquinelli et al. 2000). It was soon realised that similar sequences were scattered throughout eukaryotic genomes that were first called

The majority of human microRNAs are encoded within introns of coding or non-coding mRNAs whilst others are located exgenically, within the exons of non-coding mRNAs or

transformed than *de novo* DLBCL (Berglund et al. 2007).

lymphogenesis (Roehle et al. 2008; Lawrie et al. 2009).

lymphoid malignancy in particular (Lawrie 2008; Iorio & Croce 2009).

microRNAs in 2001 (Lee & Ambros 2001).

**3.1 MicroRNA biosynthesis and function**

**3. MicroRNAs**

apoptotic molecule BCL2. This translocation is detectable in approximately 75% of cases by traditional karyotyping techniques but in over 90% of cases measured by polymerase chain reaction (PCR) (Tsujimoto et al. 1985). Although considered to be an essential feature of FL pathology, this genetic insult appears not to be in itself sufficient to cause FL as *BCL2* transgenic mice do not readily develop lymphoma (McDonnell & Korsmeyer 1991; Strasser et al. 1993). Furthermore, the t(14;18) translocation is not an uncommon finding in normal B cells, being detectable in over 50% of healthy individuals (Roulland et al. 2006). Therefore, whilst the presence of t(14:18) is highly suggestive of FL, it is by no means diagnostic, and indeed may be found in other apparently unrelated cases of NHL including 15-30% of DLBCL cases (Iqbal et al. 2004). Furthermore about 5% of FL cases lack the t(14:18) translocation instead being characterised by a BCL6 translocation t(3;14) and displaying an almost exclusive centroblastic morphology (Jaffe et al. 2001).

FL predominantly affects adults with a median age of 59 years and a male:female ratio of 1:1.7 (Anderson et al. 1998). Most FL patients already have widespread disease at time of presentation, predominantly in the lymph nodes, but FL may also involve the spleen, bone marrow, and peripheral blood and occasionally extra-nodal sites such as the gastrointestinal tract or skin. Patients may be asymptomatic with slowly progressive lymphadenopathy or present with symptomatic complications of advancing tumour growth that require treatment. In nearly all survival studies, despite initial responsiveness to treatment, most patients relapse, and will eventually die of their disease.

#### **2.1 Histological transformation of FL tumors**

A percentage of FL patients (10-60% depending on the study) will eventually undergo high grade transformation from indolent FL to a much more aggressive tumor that is histologically indistinguishable from DLBCL, and is associated with a much poorer prognostic outcome. A recent study of 325 patients (median follow-up 15 years) found the risk of transformation to be 28% (Montoto et al. 2007). Despite the use of high dose therapy for transformed FL (tFL) cases, response rates are still lower than histologically equivalent *de novo* cases of DLBCL with a median survival of just 1.2 years. The molecular mechanisms behind this phenomenon, however, are very poorly understood and consequently the identification of at-risk patients, who might benefit from up-front high dose treatment modalities, remains one of the greatest challenges facing onco-hematologists today.

Lossos *et al* identified 671 genes that were aberrantly expressed in at least three of twelve paired biopsy samples which fell into two distinct groups; those that had *c-myc* and its target genes up-regulated and those where these genes were down-regulated (Lossos et al. 2002). Another study of five paired samples identified 36 up-regulated and 66 downregulated genes, seven of which were common with the study of Lossos *et al* (de Vos et al. 2003). Sixty-seven and 46 genes were found to be up-regulated and down-regulated respectively in a series of eleven paired samples analyzed by Elenitoba-Johnson *et al* (Elenitoba-Johnson et al. 2003). Up-regulation of p38BMAPK was confirmed immunohistochemically as it was detected in DLBCL cases but not FL or normal GC cells. Davies *et al* examined the gene expression profile of twenty paired lymphoma samples taken pre- and post-transformation (Davies et al. 2007). They found that transformation proceeded by at least two molecular pathways; one characterized by a cell proliferation signature that was associated with recurrent oncogenic abnormalities and a decrease in T cell and follicular dendritic cell genes, while the other group showed no increase in

apoptotic molecule BCL2. This translocation is detectable in approximately 75% of cases by traditional karyotyping techniques but in over 90% of cases measured by polymerase chain reaction (PCR) (Tsujimoto et al. 1985). Although considered to be an essential feature of FL pathology, this genetic insult appears not to be in itself sufficient to cause FL as *BCL2* transgenic mice do not readily develop lymphoma (McDonnell & Korsmeyer 1991; Strasser et al. 1993). Furthermore, the t(14;18) translocation is not an uncommon finding in normal B cells, being detectable in over 50% of healthy individuals (Roulland et al. 2006). Therefore, whilst the presence of t(14:18) is highly suggestive of FL, it is by no means diagnostic, and indeed may be found in other apparently unrelated cases of NHL including 15-30% of DLBCL cases (Iqbal et al. 2004). Furthermore about 5% of FL cases lack the t(14:18) translocation instead being characterised by a BCL6 translocation t(3;14) and displaying an

FL predominantly affects adults with a median age of 59 years and a male:female ratio of 1:1.7 (Anderson et al. 1998). Most FL patients already have widespread disease at time of presentation, predominantly in the lymph nodes, but FL may also involve the spleen, bone marrow, and peripheral blood and occasionally extra-nodal sites such as the gastrointestinal tract or skin. Patients may be asymptomatic with slowly progressive lymphadenopathy or present with symptomatic complications of advancing tumour growth that require treatment. In nearly all survival studies, despite initial responsiveness to

A percentage of FL patients (10-60% depending on the study) will eventually undergo high grade transformation from indolent FL to a much more aggressive tumor that is histologically indistinguishable from DLBCL, and is associated with a much poorer prognostic outcome. A recent study of 325 patients (median follow-up 15 years) found the risk of transformation to be 28% (Montoto et al. 2007). Despite the use of high dose therapy for transformed FL (tFL) cases, response rates are still lower than histologically equivalent *de novo* cases of DLBCL with a median survival of just 1.2 years. The molecular mechanisms behind this phenomenon, however, are very poorly understood and consequently the identification of at-risk patients, who might benefit from up-front high dose treatment

modalities, remains one of the greatest challenges facing onco-hematologists today.

Lossos *et al* identified 671 genes that were aberrantly expressed in at least three of twelve paired biopsy samples which fell into two distinct groups; those that had *c-myc* and its target genes up-regulated and those where these genes were down-regulated (Lossos et al. 2002). Another study of five paired samples identified 36 up-regulated and 66 downregulated genes, seven of which were common with the study of Lossos *et al* (de Vos et al. 2003). Sixty-seven and 46 genes were found to be up-regulated and down-regulated respectively in a series of eleven paired samples analyzed by Elenitoba-Johnson *et al* (Elenitoba-Johnson et al. 2003). Up-regulation of p38BMAPK was confirmed immunohistochemically as it was detected in DLBCL cases but not FL or normal GC cells. Davies *et al* examined the gene expression profile of twenty paired lymphoma samples taken pre- and post-transformation (Davies et al. 2007). They found that transformation proceeded by at least two molecular pathways; one characterized by a cell proliferation signature that was associated with recurrent oncogenic abnormalities and a decrease in T cell and follicular dendritic cell genes, while the other group showed no increase in

almost exclusive centroblastic morphology (Jaffe et al. 2001).

**2.1 Histological transformation of FL tumors**

treatment, most patients relapse, and will eventually die of their disease.

proliferation genes and followed an as yet undetermined route. In contrast a gene expression study of non-paired patients (24 FL patients who underwent transformation, 22 FL patients without transformation (after 7 years) and 24 DLBCL patients who had previously transformed from FL) found that gene expression was too heterogeneous to reliably predict transformation (Glas et al. 2007). They did however report a correlation by immunohistochemistry with the spatial distribution to neoplastic follicles and the activation of CD4+ T cells and specifically T-helper 1 cells (*P*>0.05). They did not find any correlation with other infiltrating cell populations including CD68+ macrophages or regulatory T cells.

Additionally, genomic alterations have been demonstrated to be associated with transformation of FL. The acquisition of novel mutations in PIM-1, PAX-5, RhoH/TTF and c-MYC genes, due to aberrant somatic hypermutation, was found in 5/9 cases that had undergone transformation (Rossi et al. 2006). Genomic aberrations were found to be more common in transformed cases of DLBCL than non-transformed FL and the alterations -6q16-21 and +7pter-q22 were only found in transformed DLBCL but not in follicular lymphoma whereas -4q13-21 was more common in transformed than *de novo* DLBCL (Berglund et al. 2007).

However, despite intensive research the molecular basis for transformation in FL patients remains largely unknown. Recently, ourselves and others, have raised the possibility that microRNAs may be important factors in both FL transformation and antecedent FL lymphogenesis (Roehle et al. 2008; Lawrie et al. 2009).

### **3. MicroRNAs**

MicroRNAs are a recently discovered class of naturally occurring short non-coding RNA molecules that regulate eukaryotic gene expression post-transcriptionally. There are now more than 900 human microRNAs that have been identified through cloning and/or sequence analysis (miRBase- (Griffiths-Jones et al. 2006)), and it is believed some 60% of all human genes are a target for microRNA regulation (Friedman et al. 2009). MicroRNAs have been shown to play key regulatory roles in virtually every aspect of biology including developmental timing, cell differentiation, apoptosis, cell proliferation, metabolism organ development, and hematopoiesis (Kim 2005). The potential importance of microRNAs in cancer is implied by the finding that the majority of human microRNAs are located at cancer-associated genomic regions (Calin et al. 2004), and there is now overwhelming evidence that dysfunctional expression of microRNAs is a common, if not ubiquitous, feature of cancer in general and lymphoid malignancy in particular (Lawrie 2008; Iorio & Croce 2009).

Despite the fundamental role that microRNAs appear to play in biology, these molecules were unknown to the scientific world until 1993 when *lin-4*, a *C. elegans* developmental regulator was identified (Lee et al. 1993; Wightman et al. 1993). The significance of this finding was not however realised until seven years later when another worm microRNA, *let-7* was discovered (Reinhart et al. 2000). Unlike *lin-4*, the sequence of *let-7* was found to be highly conserved in almost all organisms (Pasquinelli et al. 2000). It was soon realised that similar sequences were scattered throughout eukaryotic genomes that were first called microRNAs in 2001 (Lee & Ambros 2001).

#### **3.1 MicroRNA biosynthesis and function**

The majority of human microRNAs are encoded within introns of coding or non-coding mRNAs whilst others are located exgenically, within the exons of non-coding mRNAs or

MicroRNA Expression in Follicular Lymphoma 397

exported from the nucleus to the cytoplasm by Exportin5 in a Ran-GTP dependent manner (Zeng 2006). The cytoplasmic pre-microRNA is further cleaved to form an asymmetric duplex intermediate (microRNA: microRNA\*) by Dicer, another RNaseIII-type enzyme. Similar to Drosha, cofactors such as TRBP and PACT (in humans) are necessary for Dicer activity (Lee et al. 2006). The microRNA: microRNA\* duplex is in turn loaded into the miRISC complex in which Argonaut (Ago) proteins appear to be the key effector molecules. The strand that becomes the active mature microRNA appears to be dependent upon which has the lowest free energy 5' end and is retained by the miRISC complex whilst the other strand is usually degraded by an unknown nuclease (Khvorova et al. 2003; Schwarz et al.

The loaded miRISC is guided by the mature microRNA sequence (19-24 nucleotide) to partially complementary sequences within the 3'UTR (and probably coding sequences and 5'UTR as well) of the target mRNA, leading to inhibition of translation, transcript degradation, or both (Lawrie 2007; Lytle et al. 2007). Although repression of translation without mRNA degradation was originally believed to be the *modus operandi* of animal microRNAs, the situation appears to be more complex than previously thought, as there is now compelling evidence that microRNAs also effect transcriptional levels through deadenylation and/or degradation (Giraldez et al. 2006) and may even positively affect translation in some instances (Vasudevan et al. 2007). How translational repression occurs remains unclear. It has been suggested that mRNA bound to the microRNA-miRISC complex may be sequestered away from the translational machinery in P-bodies that additionally act in concert with enzymes to remove the 5'-cap hence preventing translation (Liu et al. 2005; Sen & Blau 2005). Alternatively it has been suggested that microRNAs may

The following experimental details were taken in part from previously published research (Lawrie, CH et al., 2009). The only other study, as far we are aware, that considers microRNA expression in FL, was by Roehle *et al* which although it included 46 FL samples, only measured levels of 153 microRNA probes (compared with 464 microRNA probes in

Formalin-fixed paraffin-embedded (FFPE) biopsy samples from 98 patients were obtained from the Pathology Department of the John Radcliffe Hospital, Oxford, UK. Eighty patients were diagnosed histologically and clinically as having DLBCL; 64 *de novo* (DLBCL-de novo) and 16 transformed cases with previously diagnosed FL (DLBCL-t). Of the 18 cases of FL used in this study, seven subsequently underwent high grade transformation (FL-t) with a median time to transformation of 24 months (range 10-96 months) from initial diagnosis. The remaining 11 FL cases (FL-nt) had no recorded transformation events (median follow-up time 60 months; range 52-132 months). The FL-t and DLBCL-t samples were not paired. All FL cases were grade 1 or 2 at time of original

prevent recognition of the 5'cap by translation factors (Pillai et al. 2005).

this study), and did not consider FL transformation (Roehle et al. 2008).

**4. MicroRNA expression in FL**

**4.1 Materials and methods**

**4.1.1 Patient material**

2003).

within the 3'UTR sequence of mRNA (Rodriguez et al. 2004). MicroRNAs are transcribed as 5'-capped large polyadenylated transcripts (pri-microRNA) primarily in a Pol II-dependent manner (Figure 1), although the involvement of Pol-III transcription has also been postulated for microRNAs encoded within Alu repeat sequences (Borchert et al. 2006). Approximately 40% of human microRNAs are co-transcribed as clusters encoding up to eight distinct microRNA sequences in a single pri-microRNA transcript (Altuvia et al. 2005; Hertel et al. 2006). Pri-microRNAs are cleaved within the nucleus by Drosha, an RNaseIIItype nuclease, to form 60-70 nucleotide hairpin structures (pre-microRNA). Drosha by itself possesses little enzymatic activity and requires the cofactor DiGeorge syndrome critical region 8 gene (DGCR8) in humans (Pasha in *Drosophila*) to form the so-called microprocessor complex (Yeom et al. 2006). Once produced, the pre-microRNAs are

Fig. 1. **Schematic diagram of microRNA biosynthesis and function in animal cells.** PrimicroRNA precursor is transcribed in Pol-II dependent manner and then cleaved by microprocessor complex (Drosha/DGCR8) to form hairpin-structure pre-microRNA. PremicroRNAs are exported from the nucleus by exportin-5 in a RAN-GTP dependent manner where they are cleaved into an asymmetric duplex by action of Dicer and accessory proteins. The mature microRNA is loaded into the miRISC complex which binds to cognate 3'UTR sequence of target mRNA resulting in either degradation of mRNA, or to blockage of translation without mRNA degradation.

within the 3'UTR sequence of mRNA (Rodriguez et al. 2004). MicroRNAs are transcribed as 5'-capped large polyadenylated transcripts (pri-microRNA) primarily in a Pol II-dependent manner (Figure 1), although the involvement of Pol-III transcription has also been postulated for microRNAs encoded within Alu repeat sequences (Borchert et al. 2006). Approximately 40% of human microRNAs are co-transcribed as clusters encoding up to eight distinct microRNA sequences in a single pri-microRNA transcript (Altuvia et al. 2005; Hertel et al. 2006). Pri-microRNAs are cleaved within the nucleus by Drosha, an RNaseIIItype nuclease, to form 60-70 nucleotide hairpin structures (pre-microRNA). Drosha by itself possesses little enzymatic activity and requires the cofactor DiGeorge syndrome critical region 8 gene (DGCR8) in humans (Pasha in *Drosophila*) to form the so-called microprocessor complex (Yeom et al. 2006). Once produced, the pre-microRNAs are

Fig. 1. **Schematic diagram of microRNA biosynthesis and function in animal cells.** PrimicroRNA precursor is transcribed in Pol-II dependent manner and then cleaved by microprocessor complex (Drosha/DGCR8) to form hairpin-structure pre-microRNA. PremicroRNAs are exported from the nucleus by exportin-5 in a RAN-GTP dependent manner where they are cleaved into an asymmetric duplex by action of Dicer and accessory proteins. The mature microRNA is loaded into the miRISC complex which binds to cognate 3'UTR sequence of target mRNA resulting in either degradation of mRNA, or to blockage of

translation without mRNA degradation.

exported from the nucleus to the cytoplasm by Exportin5 in a Ran-GTP dependent manner (Zeng 2006). The cytoplasmic pre-microRNA is further cleaved to form an asymmetric duplex intermediate (microRNA: microRNA\*) by Dicer, another RNaseIII-type enzyme. Similar to Drosha, cofactors such as TRBP and PACT (in humans) are necessary for Dicer activity (Lee et al. 2006). The microRNA: microRNA\* duplex is in turn loaded into the miRISC complex in which Argonaut (Ago) proteins appear to be the key effector molecules. The strand that becomes the active mature microRNA appears to be dependent upon which has the lowest free energy 5' end and is retained by the miRISC complex whilst the other strand is usually degraded by an unknown nuclease (Khvorova et al. 2003; Schwarz et al. 2003).

The loaded miRISC is guided by the mature microRNA sequence (19-24 nucleotide) to partially complementary sequences within the 3'UTR (and probably coding sequences and 5'UTR as well) of the target mRNA, leading to inhibition of translation, transcript degradation, or both (Lawrie 2007; Lytle et al. 2007). Although repression of translation without mRNA degradation was originally believed to be the *modus operandi* of animal microRNAs, the situation appears to be more complex than previously thought, as there is now compelling evidence that microRNAs also effect transcriptional levels through deadenylation and/or degradation (Giraldez et al. 2006) and may even positively affect translation in some instances (Vasudevan et al. 2007). How translational repression occurs remains unclear. It has been suggested that mRNA bound to the microRNA-miRISC complex may be sequestered away from the translational machinery in P-bodies that additionally act in concert with enzymes to remove the 5'-cap hence preventing translation (Liu et al. 2005; Sen & Blau 2005). Alternatively it has been suggested that microRNAs may prevent recognition of the 5'cap by translation factors (Pillai et al. 2005).

### **4. MicroRNA expression in FL**

The following experimental details were taken in part from previously published research (Lawrie, CH et al., 2009). The only other study, as far we are aware, that considers microRNA expression in FL, was by Roehle *et al* which although it included 46 FL samples, only measured levels of 153 microRNA probes (compared with 464 microRNA probes in this study), and did not consider FL transformation (Roehle et al. 2008).

#### **4.1 Materials and methods**

#### **4.1.1 Patient material**

Formalin-fixed paraffin-embedded (FFPE) biopsy samples from 98 patients were obtained from the Pathology Department of the John Radcliffe Hospital, Oxford, UK. Eighty patients were diagnosed histologically and clinically as having DLBCL; 64 *de novo* (DLBCL-de novo) and 16 transformed cases with previously diagnosed FL (DLBCL-t). Of the 18 cases of FL used in this study, seven subsequently underwent high grade transformation (FL-t) with a median time to transformation of 24 months (range 10-96 months) from initial diagnosis. The remaining 11 FL cases (FL-nt) had no recorded transformation events (median follow-up time 60 months; range 52-132 months). The FL-t and DLBCL-t samples were not paired. All FL cases were grade 1 or 2 at time of original

MicroRNA Expression in Follicular Lymphoma 399

**microRNA** *P* **value Up Fold change**

Table 1. MicroRNAs differentially expressed (*P* < 0.05) between DLBCL (DLBCL-de novo

and DLBCL-t) and FL (FL-nt and FL-t) diagnoses.

*hsa-miR-200c* 8.20E-08 DLBCL 9.39 *hsa-miR-518a* 1.23E-03 DLBCL 3.15 *hsa-miR-638* 8.05E-04 DLBCL 3.09 *hsa-miR-205* 4.36E-02 DLBCL 2.85 *hsa-miR-223* 1.42E-02 DLBCL 2.75 *hsa-miR-573* 2.79E-02 DLBCL 2.35 *hsa-miR-135b* 3.83E-02 DLBCL 1.63 *hsa-miR-133a* 8.65E-03 DLBCL 1.38 *hsa-miR-135a* 3.72E-02 DLBCL 1.38 *hsa-miR-451* 3.67E-03 DLBCL 1.38 *hsa-miR-27b* 2.12E-06 DLBCL 1.21 *hsa-miR-27a* 4.62E-07 DLBCL 1.13 *hsa-miR-18b* 1.03E-02 DLBCL 0.93 *hsa-miR-199b* 3.23E-03 DLBCL 0.83 *hsa-miR-19a* 1.20E-02 DLBCL 0.80 *hsa-miR-210* 1.10E-02 DLBCL 0.75 *hsa-miR-19b* 7.60E-04 DLBCL 0.75 *hsa-miR-99a* 9.00E-05 DLBCL 0.72 *hsa-miR-100* 1.07E-02 DLBCL 0.51 *hsa-miR-361* 3.57E-02 FL 0.58 *hsa-miR-29c* 3.07E-02 FL 0.63 *hsa-miR-26a* 8.85E-03 FL 0.73 *hsa-miR-29b* 4.22E-03 FL 0.76 *hsa-miR-26b* 5.50E-03 FL 1.04 *hsa-miR-655* 4.76E-02 FL 2.32 *hsa-miR-10b* 3.10E-02 FL 2.38 *hsa-miR-634* 1.19E-02 FL 2.41 *hsa-miR-593* 3.30E-02 FL 2.43 *hsa-miR-28* 1.47E-02 FL 2.49 *hsa-miR-150* 1.45E-03 FL 3.37

diagnosis. All samples were collected at time of initial diagnosis (i.e. prior to treatment) with the exception of DLBCL-t cases. Samples had >80% of tumor cells as determined by hematoxylin and eosin staining (not shown). Relevant ethical permission was obtained for the use of all samples.

### **4.1.2 RNA purification and microarray analysis**

Total RNA was purified from four x 20 µm FFPE sections using the Recoverall kit from Ambion (Huntington, UK) in accordance with the manufacturers' instructions. RNA (3 µg) were labeled and hybridized to µRNA microarrays as previously described (Lawrie et al. 2008) using tonsillar material (pooled from twelve healthy individuals) as a common reference in a dye-balanced design.

Image analysis was carried out with BlueFuse software (BlueGnome, Cambridge, UK). Raw image data were global median-normalized within arrays and normalized between arrays using the LIMMA package (Smyth & Speed 2003). The normalized log ratios (average of four replicates per probe) were used for subsequent analysis in Genespring 7.2 (Agilent Technologies, CA, US). ANOVA analysis was used to identify microRNAs differentially expressed between sample types and *P* values were adjusted using the Benjamini-Hodgberg correction method. Differentially expressed genes were tested for their ability to predict sample class using the leave-one-out cross-validation support vector machine (SVM) function in Genespring.

### **4.2 Results & discussion**

### **4.2.1 MicroRNA expression is distinct between DLBCL and FL**

In order to investigate differences in microRNA expression between FL and DLBCL samples, and because *de novo* and transformed DLBCL are indistinguishable histologically, we initially compared expression in all DLBCL cases (n = 80) with that of all FL cases (n = 18). Thirty microRNAs were found to be differentially expressed (P < 0.05) (Table 1). Expression values of these microRNAs correctly predicted 95/97 (98%) of cases as DLBCL or FL by SVM, and clustered the cases distinctly (Fig. 2A).

The study by Roehle *et al* identified 10 microRNAs that were differentially expressed between FL and DLBCL cases (Roehle et al. 2008). Only two of these microRNAs (*miR-150* and *miR-135a*) were found to be differentially expressed (*P* < 0.05) in our patient cohort, although another two microRNAs, *miR-92* and *miR-125b*, had *P* values of < 0.1. These 10 microRNAs correctly predicted 74/97 (76%) of cases according to diagnosis.

Roehle's study, however, compared *de novo* cases of DLBCL with FL cases that did not undergo subsequent transformation. Therefore, in order to compare the data directly we used the same sample types (64 DLBCL-de novo and 11 FL-nt cases) to re-analyze the data. This resulted in 26 differentially expressed (*P* < 0.05) microRNAs (Table 2), 14 of which were also present in the previous list (Table 1). These microRNAs correctly predicted 73/75 (97%) of cases in this cohort (c.f. 60/75 (80%) with the 10 microRNA signature (Roehle et al. 2008)) and 92/97 (95%) of cases in the extended cohort. Again, the two sets of samples were found to cluster distinctly using the 26-microRNA signature (Fig. 2B).

diagnosis. All samples were collected at time of initial diagnosis (i.e. prior to treatment) with the exception of DLBCL-t cases. Samples had >80% of tumor cells as determined by hematoxylin and eosin staining (not shown). Relevant ethical permission was obtained for

Total RNA was purified from four x 20 µm FFPE sections using the Recoverall kit from Ambion (Huntington, UK) in accordance with the manufacturers' instructions. RNA (3 µg) were labeled and hybridized to µRNA microarrays as previously described (Lawrie et al. 2008) using tonsillar material (pooled from twelve healthy individuals) as a common

Image analysis was carried out with BlueFuse software (BlueGnome, Cambridge, UK). Raw image data were global median-normalized within arrays and normalized between arrays using the LIMMA package (Smyth & Speed 2003). The normalized log ratios (average of four replicates per probe) were used for subsequent analysis in Genespring 7.2 (Agilent Technologies, CA, US). ANOVA analysis was used to identify microRNAs differentially expressed between sample types and *P* values were adjusted using the Benjamini-Hodgberg correction method. Differentially expressed genes were tested for their ability to predict sample class using the leave-one-out cross-validation support vector machine (SVM)

In order to investigate differences in microRNA expression between FL and DLBCL samples, and because *de novo* and transformed DLBCL are indistinguishable histologically, we initially compared expression in all DLBCL cases (n = 80) with that of all FL cases (n = 18). Thirty microRNAs were found to be differentially expressed (P < 0.05) (Table 1). Expression values of these microRNAs correctly predicted 95/97 (98%) of cases as DLBCL

The study by Roehle *et al* identified 10 microRNAs that were differentially expressed between FL and DLBCL cases (Roehle et al. 2008). Only two of these microRNAs (*miR-150* and *miR-135a*) were found to be differentially expressed (*P* < 0.05) in our patient cohort, although another two microRNAs, *miR-92* and *miR-125b*, had *P* values of < 0.1. These 10