1. Reactive lymphoid infiltrate versus extranodal marginal zone B-cell lymphoma of the gastrointestinal tract
   
   
2. Is there large cell transformation in extranodal marginal zone B-cell lymphoma?
   
   
3. Problems in classification of small B-cell lymphoma of the gastrointestinal tract
   
   
4. Problems in classification of T or NK cell lymphomas of the small bowel
   
   
5. Benign lymphoepithelial lesion versus Kuttner tumor versus extranodal marginal zone B-cell lymphoma of salivary gland

Reactive Lymphoid Infiltrate Versus
Extranodal Marginal Zone B-cell Lymphoma of the Gastrointestinal Tract

The Problem
When a mucosal biopsy of the gastrointestinal tract shows a heavy infiltrate of small lymphoid cells, the differential diagnosis between lymphoma and reactive lymphoid hyperplasia will naturally arise. [1] A diagnosis of lymphoma is favored if there is an extensive destructive lymphoid infiltrate with many areas totally lacking mucosal glands. Cases in which the lymphoid infiltrate is only patchy and does not fill up or expand the spaces between the glands are more likely to be reactive. In the older literature, the presence of reactive lymphoid follicles is regarded to favor a benign process; this is not true. Reactive lymphoid follicles are indeed frequently found in extranodal marginal zone B-cell lymphoma of MALT. [2, 3, 4]

Morphologic Clues
Lymphomatous infiltrate tends to be more destructive, with extensive permeation between the preexisting glands.

Presence of more than a few convincing lymphoepithelial lesions strongly favors a diagnosis of lymphoma, although lymphoepithelial lesions per se are not specific, and can be seen in florid Helicobacter gastritis or overlying a reactive lymphoid follicle ("Peyer's patch"). Lymphoepithelial lesions are defined as glandular structures expanded or distorted by groups of more than 3 lymphoid cells. The mere presence of a few scattered small lymphoid cells among the epithelial cells is insufficient for such a designation (in fact these lymphoid cells are often of T lineage). [5]

Isolated or small groups of oncocytic epithelial cells represent residual destroyed crypt cells, and represent burned-out lymphoepithelial lesions. These structures may be misinterpreted as signet ring cell carcinoma.

Assess the cytologic features of the small lymphoid cells. Presence of a significant number of small cleaved cells (larger than small lymphocytes, and showing more open chromatin and irregular foldings of nuclei) or monocytoid B cells (medium-sized cells with clear cytoplasm) supports a diagnosis of lymphoma. However, some lymphomas can be composed of lymphoid cells with minimal atypia, while the lymphocytes in reactive lymphoid infiltrate can show some irregular foldings of the nuclei.

Assess the plasma cells. Presence of Dutcher bodies or intranuclear inclusions in more than a few plasma cells or frequent cytoplasmic crystalline inclusions (rod-shaped or rhomboid) is suggestive of lymphoma; the mere presence of some degree of immaturity or nuclear enlargement has no significance.

Monotony of lymphoid infiltrate (i.e. without admixed plasma cells) favors an interpretation of lymphoma, although presence of plasma cells is of no discriminatory value.

How can immunohistochemistry help?
In a reactive lymphoid infiltrate, B cells (CD20+) are typically organized in nodules even though lymphoid follicles may not be evident. The interfollicular zone is rich in T cells (CD3+), with only isolated or at most small groups of CD20+ B cells. If immunostaining shows an extensive (at least half of a low power field using x4 objective), diffuse, densely packed infiltrate of B cells (CD20+) with limited numbers of interspersed T cells (CD3+), this represents an aberrant immunoarchitecture supportive of a diagnosis of B-cell lymphoma. It is preferable that the CD20+ infiltrate extensively percolates between the residual glands. A diffuse, dense infiltrate of T cells (CD3+), on the other hand, is inconclusive, because this can be seen either in reactive lymphoid infiltrate or T-cell lymphoma.

Monotypic Ig (frozen or paraffin sections), if successfully demonstrated, is diagnostic of lymphoma. Coexpression of CD43 by the CD20+ cells supports a diagnosis of lymphoma, but lack of aberrant immunophenotype does not exclude this possibility.

Molecular studies
Demonstration of clonally rearranged Ig gene by PCR, or rearranged oncogene such as t(11;18(q21;q21) with API2/MALT1 or t(14;18)(q32;q21) with MALT/IgH (PCR or FISH) supports a diagnosis of lymphoma.

What to do if the special studies are inconclusive?
In cases in which the various special studies are non-contributory or give equivocal results, a label of "atypical lymphoid infiltrate of undetermined significance" can be applied. In repeat biopsy, some tissue can be sent for flow cytometric analysis, and some tissue can be frozen for demonstration of surface Ig.

Is There Large Cell Transformation in Extranodal Marginal Zone B-cell Lymphoma?

Criteria of large cell transformation
Extranodal marginal zone B-cell lymphoma of MALT can transform to a diffuse large B-cell lymphoma [use of the term "high grade MALT lymphoma" is not recommended], when the prognosis will be worsened (similar to large B-cell lymphoma without a component of extranodal marginal zone B-cell lymphoma of MALT). [1] However, the criteria for large cell transformation are not well established. [Of interest, extranodal marginal zone B-cell lymphoma carrying t(11;18) translocation practically never transforms to a large cell lymphoma.]

In the current W.H.O. classification, a component of "large B-cell lymphoma" is considered to be present when "solid or sheet-like proliferations of transformed cells are present", but an exact quantity or size criterion (such as minimum size to qualify for "sheet") is not available. [2, 6]

Nonetheless, sometimes there is an increase of large cells intimately intermingled with the small cells. It appears that a designation such as "extranodal marginal zone B-cell lymphoma with increased large cells" is justified when the large cells account for >5% of the neoplastic cells. The NHL Classification Project shows that this will confer a slightly worse outcome compared with conventional extranodal marginal zone B-cell lymphoma. [7] An earlier study by de Jong et al also similarly showed a worse prognosis for the group with 1-10% large cells. [8]

Difficulties in distinction between marginal zone B-cell lymphoma and large B-cell lymphoma
Another difficult issue is that sometimes most of the neoplastic cells appear medium-sized (with irregularly folded nuclei and fairly open chromatin), rendering it difficult to determine whether the lymphoma represents an extranodal marginal zone B-cell lymphoma of MALT or a large B-cell lymphoma. There is no easy solution to this problem. Immunostaining for Ki67 may help; if the index is over 30-40%, the lymphoma is probably best considered a large cell lymphoma.

Problems in Classification of Small B Cell Lymphomas of the Gastrointestinal Tract

The Problem
Not all small B-cell lymphomas of the gastrointestinal tract are extranodal marginal zone B-cell lymphomas of MALT. Other small B-cell lymphomas can also involve the gastrointestinal tract.

Proper classification is important, because different types of small B-cell lymphomas have very different clinical evolution. Extranodal marginal zone B-cell lymphoma of MALT is often localized and indolent, with excellent prognosis; while mantle cell lymphoma is often disseminated and associated with poor response to therapy and a highly unfavorable prognosis.

Classification of diffuse small B cell lymphomas can be very difficult, particularly in small biopsies, where the full architectural growth pattern cannot be appreciated and the compression/crush artifacts render cytologic features difficult to assess. To complicate matters, there is marked cytologic overlap among the small B cell lymphomas. The centrocyte-like cells of extranodal marginal zone B-cell lymphoma of MALT are difficult to distinguish from the neoplastic cells of mantle cell lymphoma, or even B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL), since the lymphoma nuclei in all these types of lymphoma can be either round or irregular.

Clinical relevance of a diagnosis of extranodal marginal zone B-cell lymphoma of MALT?
The importance of recognizing extranodal marginal zone B-cell lymphoma of MALT type relates to the following:

Usually localized disease Highly favorable prognosis (disease may be curable by locoregional therapy) May involve several mucosal sites synchronously or metachronously (due to "homing" properties of the lymphoma cells?) Thus biologically very different from other small B-cell lymphomas (which often present with disseminated disease)

Gastric extranodal marginal zone B-cell lymphoma of MALT shows a strong association with Helicobacter pylori: [9, 10]

70-80% of patients respond to anti-Helicobacter therapy; the responders usually have superficial (non-myoinvasive) tumors Tumors showing t(11;18) translocation resulting in API2/MALT1 fusion usually do not respond to anti-Helicobacter therapy [11] Tumors showing large cell transformation usually (although not invariably) do not respond to anti-Helicobacter therapy; therefore multiple biopsies should be taken from the tumor to ascertain lack of large cell transformation before embarking on anti-Helicobacter therapy

Morphologic clues
No single histologic feature is pathognomonic of extranodal marginal zone B-cell lymphoma of MALT. A combination of features has to be considered to assign a lymphoma to this category.

Morphologic features suggestive of extranodal marginal zone B-cell lymphoma are:

Prominent lymphoepithelial lesions, although a minor degree of infiltration of epithelial structures by lymphoid cells or rare lymphoepithelial lesions can be seen in other lymphoma types. Failure to identify lymphoepithelial lesions however does not exclude this diagnosis. Cells with an appreciate amount of clear cytoplasm (monocytoid B-cell appearance). Reactive lymphoid follicles are frequently interspersed.

Morphologic features suggestive of mantle cell lymphoma are:

Monotonous lymphoid population, especially if interspersed cells with "naked" nuclei (follicular dendritic cells) can be identified. Presence of areas of mantle zone growth pattern. Solitary epithelioid histiocytes are commonly dispersed among the lymphoid infiltrate. Total lack of lymphoepithelial lesions [This diagnosis in particular has to be suspected when there are no lymphoepithelial lesions.]

Immunohistochemical evaluation
Immunohistochemical studies are very helpful for distinguishing the various types of small B-cell lymphomas, although the results can occasionally be inconclusive because individual cases may deviate from the classical immunophenotype.

LYMPHOMA TYPE	CD5	CD23	IgD	Cyclin D1	CD10/Bcl-6
B-CLL or small lymphocytic lymphoma	+	+	+	-	-
Extranodal marginal zone B?cell lymphoma of MALT	-	-*	-	-	-
Mantle cell lymphoma	+	-*	+	+	-
Diffuse follicular center lymphoma, predominantly small cell	-	+/-*	-	-	+

^* Meshworks of CD23+ follicular dendritic cells present.

^# Note that bcl-2 immunostain is of no help in lymphoma classification; it is positive in all of the above four types of lymphoma.

Molecular analysis
Molecular analysis can help in selected situations. Demonstration of bcl-1 (cyclin D1; CCND1) gene rearrangement, either by Southern blot technique (positive in 50-70% of cases), PCR (positive in ~40%) or FISH (positive in most cases), strongly supports a diagnosis of mantle cell lymphoma. Presence of bcl-2 gene rearrangement supports a diagnosis of follicle center lymphoma. Demonstration of t(11;18), t(14;18) or t(1;14) suggests a diagnosis of extranodal marginal zone B-cell lymphoma.

The "Unsolvable" Problem
Proper classification may remain impossible despite thorough work-up as discussed above, and a designation such as "small B cell lymphoma, not further classifiable" is justifiable. However, if the difficulties in classification are due to the small size of the sample or technical factors such as crush artifacts or poor fixation, this should be so stated in the report; procurement of further materials may solve the problem.

Caveats in diagnosis
In poorly fixed tissues, the neoplastic cells of Burkitt lymphoma or lymphoblastic lymphoma can look deceptively small, and thus may be misdiagnosed as small cell lymphoma. It is important particularly not to miss Burkitt lymphoma – the most important clue to diagnosis is the young age of the patients.

Extramedullary myeloid tumor (granulocytic sarcoma) can also masquerade as small cell lymphoma. It might be mistaken for a T-cell lymphoma due to positive staining for CD43 and CD45RO.

Problems in Classification of T or Nk Cell Lymphomas of the Small Bowel

The Problems
On encountering a small/medium-sized/large cell lymphoma of the small bowel that is CD3+ CD20-, the main differential diagnoses include: enteropathy-type T-cell lymphoma, peripheral T-cell lymphoma unspecified, and extranodal NK/T cell lymphoma. [If an intestinal lymphoma comprises anaplastic large cells and is CD30+, it should not be called "anaplastic large cell lymphoma" if there are other features consistent with enteropathy-type T-cell lymphoma, e.g. adjacent mucosa showing villous atrophy and intraepithelial lymphocytosis.] The classification requires consideration of a constellation of features.

Enteropathy-type T-cell lymphoma
This is a tumor of intestinal intraepithelial T lymphocytes. [12, 13] It usually produces small intestinal (especially jejunal) masses or ulcers, which are often multifocal. The lesions manifest as acute intestinal perforation, obstruction or hemorrhage. There may be a short history of adult celiac disease, long history of celiac disease, history of dermatitis herpetiformis, or no history of celiac disease at all. Rarely, the lymphoma presents initially in extraintestinal sites, such as the lung and skin.

In most cases, the tumor is composed mostly of pleomorphic to anaplastic large cells, although some cases can be composed of small or medium-sized cells. Epitheliotropism is a common finding. Some cases are accompanied by an intense infiltrate of eosinophils or histiocytes, which may obscure the diagnosis. The surrounding small bowel mucosa usually shows villous atrophy, crypt hyperplasia and intraepithelial lymphocytosis, indicative of celiac disease.

Usual immunophenotype and genotype:

T-lineage markers, e.g. CD3, CD7, positive; but CD5 is often negative (even the increased intraepithelial lymphocytes in the adjacent mucosa commonly show loss of CD5 expression) CD103+ (HML-1, marker for intraepithelial T-lymphocytes) Cytotoxic molecules (such as TIA1, perforin, granzyme B) are often positive CD4 and CD8 are often negative (although some cases can be CD8+) CD30 is positive in some cases Those examples comprising a monotonous medium-sized cell population commonly express CD56 EBV is usually negative

Nasal-type NK/T cell lymphoma
The intestine is a predilection site for the occurrence of nasal-type NK/T cell lymphoma. [14] The commonest presentation is bowel perforation. Most patients have stage III or IV disease on staging, and systemic symptoms are common. The disease pursues a highly aggressive course, with poor response to treatment, early relapse and early death. The majority of patients die of disease within 6 months.

The intestine is commonly ulcerated and densely infiltrated by abnormal lymphoid cells. There are commonly large areas of necrosis and perforation. The cytologic spectrum ranges from small cells, to medium-sized cells, to large cells, to a mixture of cells. There are frequently interspersed apoptotic bodies and inflammatory cells. Angiocentric and angiodestructive growth is common. There is preferential concentration of tumor cells around and within blood vessels, with infiltration and destruction of the vessel wall.

Usual Immunobiologic features:

Most cases exhibit an immunophenotype of CD2+ sCD3/Leu4- CD56+. Surface CD3/Leu4 (fresh or frozen tissue) negative; but cytoplasmic CD3 (polyclonal antibody to CD3ε; PS1) positive CD56: positive, but other natural killer cell markers (CD16, CD57) usually negative Other T cell markers (CD4, CD5, CD7, CD8): usually negative CD43 and CD45RO: usually positive Cytotoxic molecules (perforin, granzyme B, TIA-1): positive Some cases may express CD25 or CD30 TCR genes: usually germline Epstein-Barr virus: Positive in >90% of cases in Orientals, but figure may be lower in Caucasians

Classification of CD3+ lymphoma of small bowel
Presence of prominent angiocentric-angioinvasive growth and coagulative necrosis favors a diagnosis of nasal-type NK/T cell lymphoma. To support a diagnosis of enteropathy-type T-cell lymphoma, the adjacent small bowel mucosa should show villous atrophy and intraepithelial lymphocytosis. Special studies are very important to aid in the classification.

	Enteropathy-type T-cell lymphoma	Nasal-type NK/T cell lymphoma	Peripheral T-cell lymphoma unspecified
Surface CD3 (e.g. Leu4)	Usually positive	Negative	Usually positive
CD5	Almost always negative	Negative	Variable, often positive
CD56	Variable; cases with monotonous population of medium-sized often positive	Positive	Usually negative
Cytotoxic markers	Positive	Positive	Variable
EBER	Negative	Positive	Variable
TCR gene rearrangement	Positive	Negative	Positive

Benign Lymphoepithelial Lesion Versus Kuttner Tumor Versus Extranodal Marginal Zone B-cell Lymphoma of Salivary Gland

Benign or malignant lymphoid infiltrate in salivary gland?
Benign lymphoepithelial lesion (myoepithelial sialadenitis) is a benign condition of the salivary gland characterized by heavy lymphoid cell infiltration and epimyoepithelial island formation (often with interspersed basement membrane-like material). It may or may not occur in the setting of Sjogren syndrome. On immunophenotyping, there are many CD20+ B cells, with a considerable number of CD3+ T cells are still admixed.

Extranodal marginal zone B-cell lymphoma of MALT can arise de novo, or can supervene on benign lymphoepithelial lesion. There are commonly some interspersed reactive lymphoid follicles. Lymphoepithelial lesions are often prominent. The lymphoma cells in and around the lymphoepithelial lesions often assume an appearance resembling monocytoid B cells. Plasma cell differentiation is not uncommon.

Kuttner tumor (chronic sclerosing sialadenitis) of the submandibular gland is a chronic inflammatory disease believed to result from inspissated secretion, stones or microliths, and perpetuated by ascending infection. [15, 16, 17] It presents clinically as a hard swelling indistinguishable from a tumor. [18] The histologic features vary according to stage of evolution and severity of the inflammation. [15, 17] The lobular architecture is preserved and the degree of involvement varies from lobule to lobule. In the early stages, lymphoplasmacytic infiltrate commences around the salivary ducts, followed by periductal fibrosis. Focal squamous and mucous metaplasia and proliferation of the duct epithelium follow, but epimyoepithelial islands are absent or rare. The ducts may contain inspissated secretion. The lymphocytic infiltrate and fibrosis intensify and gradually involves the whole lobule which is accompanied by atrophy of the acini. Reactive lymphoid follicles are frequently present. In the advanced stage, there is marked fibrosis and loss of parenchyma, resembling cirrhosis of the liver. [19] Kuttner tumor is underrecognized and not uncommonly misdiagnosed as benign lymphoepithelial lesion; it can be distinguished from the latter by the scarcity or lack of epimyoepithelial islands and its usually more prominent sclerosis.

Morphologic and immunohistochemical features favoring a diagnosis of extranodal marginal zone B-cell lymphoma of MALT over benign lymphoepithelial lesion or Kuttner tumor^*

Morphologic features

Immunohistochemical features

Presence of collars of clear cells (monocytoid B cell-like) around the epithelial islands; this is considered to be the earliest evidence of a supervening lymphoma [abnormal architecture] [20] "Cavitation" of epithelial islands by large clusters of lymphoid cells [invasive features] Infiltrative growth, with infiltration of the lobular fibrous septa or nerves [invasive features] Many atypical plasma cells, if present [cytologic atypia]

Demonstration of diffuse dense sheets of B cells, with few intermingled T cells [abnormal architecture] Aberrant immunophenotype of B cells e.g., co-expression of CD43 [cytologic atypia] Demonstration of light chain restriction [cytologic atypia]

^* Absence of basement membrane droplets within the epithelial units renders a diagnosis of benign lymphoepithelial lesion unlikely. Such epimyoepithelial islands are practically not seen in Kuttner tumor. On the other hand, presence of basement membrane droplets in the epithelial units can be seen in benign lymphoepithelial lesion or extranodal marginal zone B-cell lymphoma supervening on benign lymphoepithelial lesion.

^* The possibility of Kuttner tumor has to be considered only for submandibular lymphoid lesions.

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