The concept of intrafollicular neoplasia has been recently acknowledged by the 2008 “WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues” . The term “in situ” lymphoma has been provisionally adopted to define a lymphoid neoplasia with an exclusive intrafollicular growth pattern. Therefore, the concept of intrafollicular neoplasia/“in situ” lymphoma is properly applied when the neoplastic cells are localized in the “place” that is occupied by the normal counterpart of the tumor cell, without invasion of surrounding structures .
Among germinal center (GC)-derived lymphomas a type of follicular lymphoma (FL) has been described, in which t(14;18) positive and/or monoclonal B cells strongly expressing BCL2 are observed in histologically abnormal follicles in lymph node specimens from individuals without overt disease [1, 3]. In the case of “in situ” FL, the accumulation of neoplastic cells is within the follicular GC only . Early reports have proposed different designations for this type of FL including “in situ localization of FL” , “incipient FL” , and “FL of compartmentalized small cleaved center cells” .
Most “in situ” FL cases might represent FL at the earliest stage of development ; but in other cases, “in situ” FL may be associated at diagnosis with overt FL or with lymphomas other than FL. Moreover, “in situ” FL may be discovered incidentally in association with malignancies of non lymphoid origin .
Besides “in situ” FL, an intrafollicular neoplasia may be observed in other lymphomas in which the putative normal counterpart of the tumor cell is located in the GC or the mantle zone or the marginal zone of the follicle . These lymphomas include nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) , angioimmunoblastic T-cell lymphoma (AITL) showing limited paracortical involvement [9, 10], mantle cell lymphoma (MCL) , and cases of nodal marginal zone lymphoma  (see Fig. 1). In these cases, an intrafollicular pattern of growth may be either the exclusive or the dominant pattern of growth. Importantly, most patients affected by these “in situ” or early lesions present with asyntomatic and localized disease to a limited groups of peripheral lymph nodes, or to the spleen or to another extranodal site . The course of the disease is usually indolent. However, an overt lymphoma may develop with time in a sugroups of individuals. Therefore, the relevance of the topic is not restricted to the biologic meaning of these lymphomas, but extends to their proper clinical management.
This article highlights the characteristics of intrafollicular neoplasias/“in situ” lymphomas, their classification, morphology, and the immunodiagnostics along with the main open questions including the emerging impact on the management of the affected individuals.
Tables I and II and Fig. 1 propose a classification of intrafollicular neoplasia/“in situ” lymphoma according to the location of the putative normal counterpart of the tumor cells. Tables I and II include the intrafollicular neoplasia/“in situ” lymphomas acknowledged by the WHO (2008) [1, 11], along with those tumors that may exhibit an exclusive or dominant intrafollicular pattern of growth and that usually remain localized for a long period of time. These lymphomas indeed may fulfil the definition of “in situ” lymphoma. The clinical meaning of these diagnoses is reported according to the recognized clinical conditions.
Table I. Classification of Intrafollicular Neoplasia/In Situ Lymphoma: A Proposal
Location: Follicular both germinal and mantle zone
“In Situ” FL and MCL Composite Variant
Morphology and Immunodiagnostics of GC-Derived Lymphomas
FLs are derived from GC B cells and maintain the gene expression program of this stage of differentiation . “In situ” FL was originally described  as being one of the early events associated with FL development. The t(14;18)(q32;q21) translocation is the genetic hallmark and early initiating event of FL pathogenesis. The presence of this translocation within the lymph node in “in situ” FL and in the peripheral blood of healthy individuals is in keeping with the hypothesis that these conditions represent potentially sequential steps in the molecular pathogenesis leading to FL [14, 15]. However, accumulation of genomic alterations and clonal selection are clearly required for full malignant transformation and FL progression. In some cases FL “transforms” into an aggressive lymphoma resembling diffuse large B-cell lymphoma, and this transformation can be associated with a variety of oncogenic changes .
Immunohistochemically, in “in situ” FL the involved follicles show strongly positive staining for BCL2 and CD10. The BCL2+ cells are confined only to GCs and are not seen in the interfollicular region or elsewhere in the lymph node (Table III and Figs. 2 and 3). In duodenal and splenic FL the morphology and the immunophenotypic features are similar to those of nodal FL [1, 18]. The follicles in the mucosa, indeed, are uniformly positive for BCL2 and for CD10. The genetic features of duodenal FL are similar to those of nodal FLs. Duodenal FL presents as multiple small polyps, usually as an incidental finding on endoscopy performed for other reasons.
Table III. Intrafollicular Neoplasia/In Situ Lymphoma: Diagnostic Features of Germinal Center-Derived In Situ Lymphomasa
Differential diagnosis with overt FL and overt NLPHL: in overt FL and NLPHL, infiltration of the interfollicular regions and involvement of most follicles are usual. Differential diagnosis with overt AITL: in overt AITL a wide paracortical involvement is usual.
The molecular profile of AITL is characterized by overexpression of several genes characteristic of normal TFH cells, such as CXCL13, BCL6, PDCD1, CD40L, and NFATC1. Overexpression of these genes in AITL was validated by immunohistochemistry .
The intrafollicular pattern in NLPHL (Table III and Fig. 4) is usually seen in areas of partially involved lymph nodes that otherwise show the most common nodular involvement by the tumor. Importantly, the observation of this isolated pattern in lymph nodes of occasional patients with an indolent clinical course , suggest that it represents the initial infiltration of the follicle by the tumor cells. Conversely, the presence of numerous lymphocyte predominant cells (LP cells) outside the follicles predicts for progression to a diffuse pattern . This intrafollicular neoplasia in an NLPHL is likely to be an “in situ” step potentially leading to overt Hodgkin lymphoma.
Angioimmunoblastic T-cell lymphoma
In early lymph-node involvement by AITL, the neoplastic T cells preferentially occupy the B-cell follicles and immediate perifollicular area, sometimes mimicking a FL of B-cell origin (see Fig. 5) [9, 21–24]. Typically, the tumor cells of AITL have a T-helper cell phenotype expressing CD3, CD4, and frequently CD10, similar to a subset of normal GC-Th cells [23, 25] (Table III). The neoplastic CD4+ T cells represent a minority of the lymph node cell population, their detection being facilitated by the aberrant expression of CD10 (Table III).
Morphology and Immunodiagnostics of Mantle Cell-Derived B-Cell Lymphoma
Mantle cell lymphoma
Cases of “in situ” MCL are anedoctal [26, 27] with some difficulties in distinguishing between MCL with a nodular pattern and real “in situ” MCL . Although the problem of “in situ” lesions for MCL is increasingly recognized in the routine clinical practice, no clear guidelines for diagnosis of these patients have been defined so far. In “in situ” MCL, the lymphoma cells are restricted to the inner mantle zone , whereas in early involvement of overt MCL, lymphoma cells substitute the mantle zone and tend to invade the reactive GC. In cases with features intermediate between these distinct patterns, it is difficult to draw a definite line between “in situ” MCL and early involvement of overt MCL showing a mantle zone pattern [2, 28].
Morphology and Immunodiagnostics of Marginal Zone Derived B-Cell Lymphoma
Among marginal zone derived B-cell lymphomas, early lesions may be observed within the subset of nodal marginal zone lymphoma of the splenic type, with tumor cells growing inside an attenuated mantle zone and often around a residual GC. The presence of remnants of FDC networks suggests colonized follicles . Immunohistochemical studies show that the neoplastic lymphocytes are reactive with CD20 and negative for CD3 in all cases. All cases are negative for CD5, CD10, and CD23, whereas CD43 and BCL2 expression on B cells is usually present .
Open Questions on Diagnosis and Management
Main open questions regard the diagnostic approach and the clinical management of “in situ” or early lymphoma especially in otherwise healthy individuals.
(1) What is the recommended approach in detecting lymphoma cases with minimal involvement?
We usually use a selected diagnostic panel of antibodies (CD20, BCL2, BCL6, CD4, CD10, Cyclin D1, and CD23) in lymph nodes with follicular hyperplasia only when we observe an unexplained lymphadenopathy (Table III). The diagnosis of intrafollicular neoplasia/“in situ” lymphoma for FL and MCL is feasible when immunohistochemical characterization is carried out with the mentioned diagnostic panel. Molecular studies should be restricted to doubtful cases in which immunohistochemistry data are ambiguous.
For the other lymphomas (NLPHL and AITL) the diagnosis is based on characteristic histopathologic features and selected immunophenotypic criteria  (Table III). The intrafollicular pattern of growth is correctly identifiable based on immunohistochemical recognition of the CD23 positive meshwork of FDCs highlighting the GC involvement by neoplasia.
(2 and 3) How to approach and monitor these types of cases with an uncertain clinical behavior and unknown risk to progression to overt lymphoma? How to treat?
The natural history of these entities is poorly defined and appropriate management, surveillance, and follow up are still to be defined. However, since the risk for progression is not yet fully known for these early lesions, their management in otherwise healthy individual should consider a staging work-up to exclude the possible coexistence of an overt lymphoma in other sites.
The following questions are still unanswered:
(4) Are there biologic features that will identify cases that are at risk of developing overt lymphoma?
(5) What is the frequency with which “in situ” lymphomas occur?
(6) What is the frequency of concomitant systemic disease?
Finally, an important challenge is to understand whether the occurrence of an “in situ” lymphoma may confer any prognostic information when it is associated with lymphoid or non lymphoid malignancies.
Tumors that remain localized for a long period of time (for example duodenal or splenic FL and nodular NLPHL) should be included under the definition of “situ” lymphoma in cases in which the tumor cells/LP cells do not expand beyond the GC and do not infiltrate the mantle zone or the interfollicular zones. Conversely, in other cases in which tumor cells initially invade the surrounding structures the definition of early lymphoma is preferable.