Management of lymphomas has constantly evolved over the years and newer concepts are likely to emerge in the next few years with the advent of DNA gene chip technology. Research at mRNA level will require validation by imunohistochemistry (IHC), and will thus be incorporated in diagnostic work in specialized hematopathology laboratories.

Diagnosis and further classification of lymphomas is important as treatment protocols are different. Diffuse large B-cell lymphomas (DLBCL) receive Rituximab in addition to conventional CHOP. Burkitt’s and lymphoblastic lymphomas have excellent prognosis if diagnosed early. Hodgkin’s disease (HD) has ABVD protocol. Classical HD may resemble T-cell rich B-cell lymphoma which is an aggressive lymphoma and behave like DLBCL.

Lymphoma diagnosis involves a multiparameter approach and includes most importantly a meticulous H&E examination followed by IHC, cytogenetics and molecular diagnostics. Also important are history, clinical features, and laboratory parameters such as CBC, ESR, serum albumin and beta-2 microglobulin. Bone marrow examination, X-ray chest and abdominal ultrasonography are required for staging of the disease. Anterior mediastinal mass in a young individual causing superior vena cava syndrome, raises a suspicion of lymphoblastic lymphoma. Toxoplasmosis commonly involves the posterior occipital nodes. Low grade lymphomas are generally seen in elderly age group. Pediatric lymphomas mostly include HD, lymphoblastic lymphoma (LL), Burkitt’s lymphoma (BL), anaplastic large cell lymphoma (ALCL) and DLBCL.

To devise diagnostic panels for different types of lymphomas, one has to use his own expertise and the best available external evidence. The diagnosis is usually made on a good quality H&E slide applying morphologic diagnostic criteria. IHC is performed to confirm the diagnosis, for therapeutic purposes (Rituximab, alemtuzumab), and some times to differentiate between a reactive and a malignant lymphoid proliferation.

Immunophenotyping of lymphomas as B or T cell type is not straight-forward as many of them have a polymorphous population. It is important to understand different CD markers in details, and also their reaction to not only malignant lymphoid proliferations but also to normal reactive lymphoid cells. A normal lymph node has many compartments with different types of cells. All these compartments react differently to different antibodies. An example is of bcl-2 reaction in a normal lymph node where it shows positivity with mantle cells, interfollicular T cells and in T cells (CD4+) in the germinal centers.

Histology and Lymphoma Diagnosis
Many benign conditions like collagen vascular diseases and viral infections like EBV, CMV, HIV infected nodes may mimic lymphomas as is Kikuchi’s disease. Other diseases like dermatopathic lymphadenitis, Castleman’s disease may mimic Hodgkin disease. Many cases of Hodgkin’s disease may contain epithelioid cell granulomas mimicking tuberculosis. Small granulomas may be seen in toxoplasmosis.

Low magnification approach is a rule : One has to spend maximum time on scanning and low power examination of a lymph node biopsy to study colors, architectural features, size and shape of different compartments, their relationship to each other, and patterns. Maximum information is obtained on a meticulous scanning and low power examination of a well stained H&E section.
For lymphoma diagnosis, different types of patterns have been defined on the basis of architectural features, patterns and colors in lymph node pathology :

1. Microscope prerequisites : A good quality microscope with an objective scanning lens (2 X or 2.5 X) is a must. It helps us to identify patterns and relationships.

2. Architecture : Normal lymphoid architecture is best appreciated on a low power examination. Examine follicles, paracortical region, medulla, patent sinuses.

3. Recognizing cells based on colors : Various cell types and also the areas as follicles, mantle zones, marginal zones, interfollicular zones, sinuses and medulla may be recognized on the basis of colors in the low magnification.

4. Assessing cellularity : It is also best appreciated on a low power examination. Based on cellularity, the various lymphomas may be broadly classified as follows:

- Hypercellular lymphomas : Lymphomas of small cell type are hypercellular, for example, mantle cell lymphoma, small cell lymphoma (MCL), follicular lymphoma (FL), LL, and BL - all have monomorphic cells. Other hypercellular lymphoid malignancies include HD-Lymphocyte depleted (HD-LD), HD-Nodular sclerosis (HD-NS)-Type 2, acute leukemias.

- Normocellular lymphomas – HD - mixed cellularity (HD-MC), HD-NS Type 1, Nodular Lymphocytic predominant - HD (NLPHD), Marginal zone lymphoma (MZL), Peripheral T-Cell Lymphoma (PTCL).

- Hypocellular lymphomas - Angioimmunoblastic T-cell lymphoma, Histiocyte-rich B-cell lymphoma, Histiocyte-rich ALCL, HD-MC, HD-NS Type 1, MZL, PTCL.

5. Identifying pathologic processes (predominant/diffuse and focal) : Lymph node involvement may be predominant or may be focal. Thus one must identify all the areas including the capsule, sub-capsular and medullary sinuses, follicle, mantle zones, marginal zones, interfollicular areas. Focal involvement of lymphomas and acute leukemias in a lymph node biopsy is well known.

6. Patterns, their formations, and their utility : Many patterns have been described such as follicular, mantle, marginal zone, sinusoidal, interfollicular, starry sky, Lennert’s (epithelioid histiocytes), fibrous nodular, pseudo follicular. They all denote different lesions. These are best viewed by altering the intensity of the light, moving condenser, and by changing the power. Different lymphomas may have different patterns, like mantle cell lymphoma may have either diffuse, nodular or mantle zone patterns.

Although clusters of epithelioid histiocytes may be seen in HD, T-cell lymphomas, morphological features such as clusters of epithelioid cells (small granulomas) within the follicle is pathognomonic of toxoplasmosis.

Prognostic factors are important in lymphomas. We may take an example of follicular lymphoma where favorable markers include increased small vessel density, bcl6 expression, CD10 expression, while unfavorable markers include diffuse areas, increasing cytological grade, interfollicular proliferation and bcl2 expression etc. CLL has two subtypes, the type with CD38+ and high ZAP70 expression behave more aggressively.

Small cleaved lymphomas include mantle cell lymphomas, follicular lymphomas, small lymphocytic lymphomas. Small non cleaved cell lymphomas are mostly BL. Look at the apoptosis and proliferation index which is diagnostic. DLBCL is a waste basket and include centroblastic, immunoblastic and plasmablastic variants. It also includes T-cell rich B-cell lymphomas and B-cell ALCL.

Vascular proliferations are seen commonly in angioimmunoblastic T-cell lymphoma, PTCL (NOS), hyaline vascular type Castleman’s disease, and angioimmunoblastic lymphadenopathy.

Mottling pattern is in T-zone comprising of transformed lymphoid cells, histiocytes, interdigitating dendritic cells. Seen commonly in reactive nodes, viral infections, SLE, Kikuchi’s disease and rarely in mycosis fungoides. It has to be differentiated from the starry sky pattern which is due to histiocytes showing phagocytosis and is seen in viral infections such as infectious mononucleosis, and LL, BL, AML.

Immunophenotypic profile of cells of a normal lymph node

Germinal center B cells : CD45+, CD20+, CD79a+, CD5-, CD10+, Ig+ (IgG, IgM or IgA, but not IgD), Light chain+ (K/L), bcl-2-, bcl-6+, proliferating cells in dark zone highlighted by Ki67.

Mantle zone B cells : CD45+, CD20+, CD79a+, CD5+, IgD+, IgM+, IgG-, Light chain+, bcl-2+, bcl-6-.

Marginal zone B cells and monocytoid B cells : LCA+, CD20+, CD79a+, IgM+, IgD-/+, CD5-, bcl-2-, bcl-6-

Interfollicular B cells : CD45+, CD20+, CD79a+, Ig+ (IgD, IgM, IgG or IgA), K/L +, bcl-2+, bcl-6-,

Plasma cells : CD45+, CD20-, CD79a+/-, Ig+, CD38+, EMA+/-.

T cells : CD3+, CD45RO+, CD43+, mostly TCR a/b+, CD4+ or CD8+

T cells in germinal center : CD3+, CD45RO+, CD43+, CD57+, CD4+, CD8-.

NK cells : CD45+, surface CD3-, cytoplasmic CD3+, CD43+, CD45RO, CD56+, CD57+/-, Granzyme B+

Follicular dendritic cells : CD45-, CD21+, CD35+, CD23 (subpopulation) +, HLA-DR+.

Interdigitating dendritic cells : CD45+, S100+, CD68-/+, HLA-DR+, CD43+.

Langerhans cells : CD45+, S100+, CD68-/+, CD1a+, Lysozyme-/+, HLA-DR+.

Histiocytes : CD45+, S100-, CD68+, CD1a-, Lysozyme+, HLA-DR+, CD43+.

IHC and reporting of hematolymphoid malignancies

1. Hodgkin’s disease
IHC is not required in all the cases of classical HD. RS cells with a classical morphology in a polymorphous background is enough to label a case as a HD. IHC may be required when differential is with TCR-BCL, PTCL, ALCL, or may be other abnormal lymphoid proliferations.

2. Non-Hodgkin’s lymphoma
Minimum IHC panel for any suspected hematolymphoid lesion is LCA, CD20 and CD3. Additional panel for other circumstances based on morphological impression is as follows :

LL and BL are oncologic emergencies, and any delay in reporting may be disastrous.

DLBCL is of GCB and ABC subtypes. GCB-DLBL expresses CD10 and bcl6 and has a better overall survival, whereas ABC-DLBCL expresses MUM1/IRF4 and has poorer overall survival.

CLL is divided into somatically mutated (good prognosis) and unmutated (CD38+, ZAP 70+) subtypes.

PTCL-NOS is diagnosed after excluding commoner T-cell lymphomas like ALCL, T-LL.

Benign versus malignant lymphoid proliferations
Many benign conditions like viral and bacterial infections, HIV, toxoplasmosis, Kikuchi’s disease, dermatopathic lymphadenitis, sinus histiocytosis with and collagen vascular diseases may mimic a hematolymphoid malignancy. The nodes may be large in size and show a partial effacement of the architecture with a prominent follicular hyperplasia or/and paracortical T zone expansion. Follicular lysis and progressive transformation of germinal centres may be evident. Immunoblasts may be prominent both in the follicular as well as in paracortical zones and may react with LCA and CD30 stains. RS cells, unlike immunoblasts are generally LCA negative. Kikuchi’s disease may resemble DLBCL. Kimura’s disease, dermatopathic lymphadenitis and Castleman’s disease may resemble HD. Following may be of help in certain circumstances (in addition to morphology):

a) Light chain restriction: by doing kappa and lambda light chains. Excess of kappa-positive cells, or more Kappa than Lambda positive cells, at least exceeding ratio 10:1 ratio (others prefer 18:1), suggests a neoplastic proliferation.

b) Aberrant expression in B cells:
(i) Co-expression of a B-cell (CD20) marker with CD43 or CD5 (T-cell marker): It can be inferred that this is a neoplastic proliferation.
(ii) Expression of cyclin D1 (bcl-1): It supports a diagnosis of mantle cell lymphoma.

c) Aberrant expression in T cells : Cases of T-cell proliferation (CD3+ve) that extensively express markers not normally expressed by T cells (such as CD56, CD30, ALK1) or show loss of marker such as CD5 are almost certainly lymphomas. T-cell proliferation that exhibits a CD4+ CD8+ or CD4- CD8- (double positive or double negative) phenotype is suggestive of NHL.

d) Immature cell phenotype : Large number of lymphoid cells that expressing TdT or CD1a, whether of B or T cell lineage, is indicative of precursor LL.

e) Sheets of B cells : In nodal and extranodal sites with diffuse small lymphocytic proliferation, presence of diffuse sheets of B cells (CD20+) suggests a diagnosis of lymphoma.

f) CD10 & bcl-2 expression : is studied for nodular growth patterns and to differentiate Burkitt from DLBL. BL is CD10+, bcl-2 negative and Ki67 >95%.

g) CD23 stain : if uncertain as to whether there are neoplastic follicles (subtle follicles or nodules of lymphoid cells) to highlight FDC, depicting the follicular framework. To differentiate angioimmunoblastic T-cell lymphoma from PTCL (NOS), CD23 demonstrates the FDCs that occur outside the follicles

h) CD99 (Mic2) : stains lymphoblastic lymphoma, PNET & mesenchymal chondrosarcoma.
i) LCA negative lymphoma : particularly lymphoblastic, plasmablastic, ALCL, PTCL, classical HD and plasma cell neoplasms (even CD20 negative).

Reactive follicular hyperplasia versus follicular lymphoma
Reactive hyperplasia will have classical morphological features. It shows low density of follicles, preservation of nodal architecture, variation in size and shape of germinal centres, ample interfollicular tissue containing inflammatory cells, sharp demarcation of germinal centres from mantle zone, germinal centres will show polarization, brisk mitosis and tingible body macrophages. In addition we may do following :

a) bcl-2 staining : Positive staining of the germinal centers favors a diagnosis of follicular lymphoma (FL). A negative reaction, however, does not totally rule out FL (since only 75-80% of cases show positivity, and grade 3 FL may be negative for bcl-2). Reactive follicles contain many reactive CD4+ T-cells which are bcl-2 positive, resulting in a false positive interpretation.

b) Light chain restriction

c) Ki-67 (MiB1, proliferation marker) : In reactive follicles, Ki-67 will highlight large number of positive (proliferating) cells, accentuating the polarity (dark zone). In follicular lymphoma, polarity will not be seen, and the percentage of Ki-67 positive is generally low.

d) B-cell marker : Numerous CD20 positive B-cells between follicles suggest a FL.
Marginal zone (monocytoid) B-cell hyperplasia versus marginal zone B cell lymphoma (MZBL)

a) bcl-2 : Reactive monocytoid B cells are usually bcl-2 -ve, while MZBL is almost always bcl-2 +ve.
b) Immunoglobulin : Light chain restriction (plasma cells present in MZBL).

Classical HD versus NHL?
Classical HD may be confused with T-cell rich B-cell lymphoma (TCR-BCL), PTCL and ALCL. HD can also be confused with Castleman’s disease, viral infections and other reactive conditions. A panel including LCA, CD20, CD3, CD15, CD30, EMA, ALK-1, and MiB1 may be helpful. Staining is assessed on the large atypical cells. RS cells of classical HD express CD30, CD15 and EBER while are negative for LCA, CD3, EMA and ALK-1.

NLPHD (Nodular lymphocytic predominance Hodgkin disease) is a different type of HD. It shows large sized nodules with features of PTGC in association with follicular hyperplasia. PTGC with popcorn cells clinches the diagnosis of NLPHD. Tumor cells (popcorn cells/L&H cells) express LCA, CD20, EMA and bcl6 and negative for CD15, CD30 and EBER.

Bone marrow and CD30+ ALCL

ALCL cells in BM can be very subtle and difficult to recognize. CD30 and/or ALK1, is helpful.

Problems in diagnosis of plasma cell lesions
a) To differentiate benign plasma cell from a monoclonal plasma cell population, do light chain restriction.
b) Plasmablastic lymphomas (seen in HIV+ patients): resembles a poorly differentiated malignant tumor but negative for CK, EMA, S-100. HMB45, CD20. LCA may be equivocal. CD79a is seen in about 50% of cases. EMA is positive. Light chain restriction is diagnostic. Differentiating plasmacytic lesions from plasmablastic lesions is important, and morphology is supplemented with MiB1 (a proliferation marker).
c) May resemble a neuroendocrine tumor and light chain restriction may be carried out.

Table 4: Differential diagnosis of blastic lymphomas/leukemias and IHC

References:

1. Hans CP, et al., Confirmation of the molecular classification of DLBCL by immunohistochemistry using a tissue microarray. Blood, 2004 Jan 1;103(1):275-82. Epub 2003 Sep 22

2. Tumors of Hematopoietic and lymphoid tissues. Pathology and Genetics. World Health Organization. Classification of Tumors Edited by Jaffe ES, Harris NL, Stein H, Vardiman JW

3. Neoplastic Hematopathology. 2nd Edition. Lippincott Williams & Williams, Ed. Daniel Knowles