Pathology of the Lymphoid System

Pathology of the Lymphoid System Learning Objectives: Define lymphadenitis and enumerate its types. Briefly describe the morphological appearance of reactive lymph node. Describe the microscopic picture of the lymph node affected by acute & chronic inflammation. Classify tumors of the lymph node and spleen. Give simple classification of non-hodgkin's lymphoma. Briefly describe an example of each subgroup. Give an account on Hodgkin's disease with emphasis on histological classification & staging. Enumerate common causes of enlargement of the spleen. Define hypersplenism and enumerate its causes. Lymph Node: I. Non-Neoplastic Conditions: Reactive Lymphadenitis: Any immune response against foreign antigens (infectious & noninfectious inflammatory stimuli) is often associated with lymph node enlargement (lymphadenopathy). The infections that cause lymphadenitis are numerous and varied and may be acute or chronic. In most instances, the histological appearance of the nodes is entirely nonspecific i.e. different etiologies are associated with similar microscopic changes. Acute Nonspecific Lymphadenitis: Grossly: inflamed nodes are swollen & congested i.e. gray-red. Microscopically: there are large germinal centers containing numerous mitotic figures. When the cause is a pyogenic organism, a neutrophilic infiltrate is seen about the follicles and within the lymphoid sinuses. With severe infections, the centers of follicles can undergo suppurative necrosis. The overlying skin is frequently red, and penetration of the infection to the skin can produce draining sinuses. Chronic Nonspecific Lymphadenitis: This condition can assume one of three patterns, depending on the causative agent: 1. Follicular hyperplasia 2. Paracortical hyperplasia 3. Sinus histiocytosis Follicular Hyperplasia: is associated with infections or inflammatory processes that activate B cells in the B-cell areas i.e. the follicles, & thus create the follicular (or germinal center) reaction. The cells in the reactive follicles include the activated B cells (called follicular center cells), scattered phagocytic macrophages containing nuclear debris and follicular dendritic cells (function in antigen display to the B cells). Causes of follicular hyperplasia include: *Rheumatoid arthritis. *Toxoplasmosis. *Early stages of HIV infection. Paracortical Hyperplasia: The paracortex is the zone situated between the cortex and the medulla, which contains the mobile pool of T lymphocytes responsible for cell-mediated immune responses. Paracortical hyperplasia is characterized by reactive changes within the T- cell regions of the lymph node, which is reflected microscopically as expanded zones between 1

the cortical follicles. On immune activation parafollicular T-cells transform into large proliferating immunoblasts. Paracortical hyperplasia is encountered in: *Viral infections (such as EBV). *Following certain vaccinations. *Immune reactions induced by certain drugs. Sinus Histiocytosis: is characterized by distention and prominence of the lymphatic sinusoids, owing to a marked hypertrophy of lining endothelial cells and an infiltrate of macrophages. Sinus histiocytosis is often encountered in: *Lymph nodes draining cancers and may represent an immune response to the tumor or its products. Granulomatous lymphadenitis: Immune reactions are usually involved in the development of granulomas. There is often strong activation of T lymphocytes leading to macrophage activation, which can cause injury to normal tissues. Granulomatous inflammation is a distinctive pattern of chronic inflammation that is encountered in a limited number of infectious and some noninfectious conditions. The causes of granulomatous lymphadenitis include: Infections. Foreign body reactions. Malignancy. Among the infectious causes are: *Tuberculosis. *Atypical mycobacteriosis. *Sarcoidosis. *Fungal infections. *Toxoplasmosis. *Syphilis & Leprosy. *Brucellosis. Malignancy related granulomatous lymphadenitis: occurs whether the neoplasm is: *Primary (Hodgkin & nonhodgkin lymphomas), or *Secondary (metastatic carcinoma). It occurs whether the node is involved by the malignancy or not. II. Lymphoid Neoplasms: All lymphoid neoplasms have the potential to spread to lymph nodes and various tissues throughout the body, especially the liver, spleen, and bone marrow. In some cases lymphomas spill over into the peripheral blood, creating a leukemia-like picture. Conversely, leukemias of lymphoid cells, originating in the bone marrow, can infiltrate lymph nodes and other tissues, creating the histologic picture of lymphoma. Two groups of lymphomas are recognized: Hodgkin lymphoma and (HL). Non-Hodgkin lymphomas (NHL). According to the Iraqi Cancer Registry (2009); NHL ranks the 6 th, and represents 4.83% of the top ten cancers by primary site in Iraq. The behavior and treatment of Hodgkin lymphoma differ from those of most NHLs, thus making the distinction between the two of practical importance. It has been shown that NHLs is often widely disseminated at the time of diagnosis, even when the disease appears clinically localized, that is why only systemic therapies are curative. In contrast, Hodgkin lymphoma often presents at a single site and spreads in a predictable fashion to neighboring lymph node groups. For this reason, early in the disease, local therapy may be effective. The WHO has established a widely accepted classification that depends on a combination of: *Clinical. *Microscopic. *Immunophenotypic. *Genetic features (e.g., cytogenetic, molecular, presence of viral genomes, etc.). B- and T-cell tumors are composed of cells derived from specific stages of their normal differentiation pathways. The diagnosis and classification of these tumors relies heavily on tests 2

(e.g. immunohistochemistry & flow cytometry) that detect specific antigens (e.g., B-cell, T-cell markers). B- and T-cell tumors are composed of cells derived from specific stages of their normal differentiation pathways. All lymphomas are derived from a single transformed cell and thus are by definition monoclonal. Non-Hodgkin lymphomas (NHL): Precursor B- and T-Cell Lymphoblastic Leukemia/Lymphoma: These are aggressive tumors, composed of immature lymphocytes (lymphoblasts). They are microscopically indistinguishable. Both pre-b- and pre-t-lymphoblastic lymphomas usually take on the clinical appearance of an acute lymphoblastic leukemia (ALL) at some time during their course. As a group, ALLs constitute 80% of childhood leukemia, peaking in incidence at age of 4 years, with most of the cases being of pre-b-cell origin. The pre-t-cell tumors, which initially present as thymic tumors, are most common in adolescent males of between 15 and 20 years of age. Small Lymphocytic Lymphoma/Chronic Lymphocytic Leukemia (SLL/ CLL): These two disorders differ only in the extent of peripheral blood involvement. If there is peripheral blood lymphocytosis > 4000 /µl, the patient is diagnosed with chronic lymphocytic leukemia (CLL); if not, a diagnosis of small lymphocytic lymphoma (SLL) is made. Microscopic features: There is diffuse effacement of the lymph node architecture by lymphoid cells. The predominant cells are compact, monomorphic, small, resting lymphocytes with dark-staining round nuclei & scanty cytoplasm. There are, in addition, foci of mitotically active larger cells; these aggregated to form proliferation centers. The latter are pathognomonic for CLL/SLL. CLL/SLL is a neoplasm of mature B cells expressing pan-bcell markers such as CD19, CD20. In addition to the lymph nodes, the bone marrow is involved in almost all cases; this is associated with peripheral absolute lymphocytosis. Generalized lymphadenopathy and hepatosplenomegaly are present in > 50% of cases. CLL/SLL tends to transform to more aggressive lymphoid malignancies. Follicular Lymphomas: The tumor likely arises from germinal center B cells. The bone marrow is almost always involved at the time of diagnosis. Microscopic features: The native lymph node architecture is effaced by nodular (follicular) aggregates of proliferating lymphoid cells. Most commonly, the predominant neoplastic cells are "centrocyte-like" cells; these are slightly larger than resting lymphocytes & have "cleaved" nuclei due to prominent indentations and linear infoldings of the nuclear membrane. The small, cleaved cells are mixed with variable numbers of larger "centroblast-like" cells that have vesicular chromatin, several nucleoli, and moderate amounts of cytoplasm. These tumors express the pan-b-cell markers. The majority of tumors have a characteristic t(14;18) translocation. This translocation fuses the BCL2 gene to the IgH locus on chromosome 14 and leads to the inappropriate expression of BCL2 protein, which functions to prevent apoptosis. Diffuse Large B-Cell Lymphoma (DLBCL): this is the most important type of NHL lymphoma in adults (50% of adult NHL). Microscopic features: The nuclei of the neoplastic B cells are large (3-4 times the size of resting lymphocytes). There are two morphologic variants of DLBCLs: A. Centroblastic: the cells have round, or cleaved nuclear contours & several distinct nucleoli, and moderate amounts of pale cytoplasm. 3

B. Immunoblastic: the cells have a large round or multilobulated nucleus, one or two centrally placed prominent nucleoli, and abundant cytoplasm. The cells express pan-b-cell antigens. Approximately 30% of tumors have a t(14;18) translocation involving the BCL2 gene. Such tumors may represent "transformed" follicular lymphomas. Burkitt Lymphoma: is endemic in some parts of Africa and sporadic elsewhere. Both the African and nonendemic forms are identical, although there are clinical and virologic differences. *In endemic areas: tumor cells in almost all patients carry EBV genome. In such endemic areas concomitant (endemic) malaria (or other infections) impairs immune competence, allowing sustained B-cell proliferation. Lymphoma cells emerge only when additional mutations, such as the t(8;14) translocation. This involves MYC proto-oncogene on chromosome 8. The latter becomes activated to MYC oncogene as a result of fusion with the IgH gene on chromosomes 14. The net result is the dysregulation and overexpression of the MYC protein. *In nonendemic areas: 80% of tumors DO NOT harbor the EBV genome, but all tumors possess the specific t(8 ; 14) translocation. This observation suggests that, although non- African Burkitt lymphomas are triggered by mechanisms other than EBV, they develop cancer by similar pathways. Microscopic features: The tumor cells are uniform and intermediate in size with round nuclei containing 2 to 5 prominent nucleoli. There is a moderate amount of basophilic cytoplasm, which on cytological smears often contains small lipid vacuoles. A high mitotic rate is very characteristic of this tumor, as is cell death. The latter accounts for the presence of numerous tissue macrophages containing ingested nuclear debris. Because these benign macrophages with their pale cytoplasm are scattered within a blue background (of lymphoma cells) a "starry sky" pattern is thus created. These B-cell tumors express surface IgM, κ or λ light chain, and the pan-b-cell markers. Mantle Cell Lymphomas: are composed of B cells that resemble cells in the mantle zone of normal lymphoid follicles. They involve lymph nodes in a diffuse or vaguely nodular pattern. The tumor cells are usually slightly larger than normal lymphocytes and have an irregular nucleus. The bone marrow is involved in the majority of cases, and about 20% of patients have peripheral blood involvement. One unexplained but characteristic tendency is the frequent involvement of the gastrointestinal tract. Hodgkin Lymphoma (HL): encompasses a distinctive group of neoplasms that arise almost invariably in a single lymph node or chain of lymph nodes and spread characteristically in a stepwise fashion to the anatomically contiguous nodes. HL accounts for 30% of all lymphomas. Classification: HL has been classified into: 1. Classical HLs. 2. Nodular lymphocyte predominant HL. Classical Hodgkin's lymphoma are subclassified into 4 pathologic subtypes based upon Reed-Sternberg cell morphology and the composition of the reactive cell infiltrate seen in the lymph node biopsy specimen. Microscopic features: The characteristic microscopic feature is the presence of distinctive neoplastic giant cells called Reed-Sternberg (RS) cells in a reactive background of prominent inflammatory response composed of a variable number of lymphocytes, macrophages, eosinophils, plasma cells and fibroblasts. RS cell is a large cell (15-45 μm in diameter) with an enlarged bi- or multilobated nucleus, prominent nucleoli, and abundant, usually slightly eosinophilic cytoplasm. Particularly 4

characteristic are cells with two mirror-image nuclei or nuclear lobes, each containing a large acidophilic nucleolus surrounded by a distinctive clear zone; together they impart an owl-eye appearance. The nuclear membrane is thick. Also present are large cells of a similar appearance but with a single round nucleus and a very large nucleolus, designated mononuclear Hodgkin cells. Immunophenotype: Hodgkin and RS cells are CD30 positive in nearly all cases, and CD15 positive in most. In 30-40% of cases CD20 may be detected, usually of varied intensity and in a minority of Hodgkin and RS cells. Classical HL: Under this heading are the following subtypes: Nodular Sclerosis Hodgkin Lymphoma: is the most common form. It is equally frequent in men and women and has a striking tendency to involve the lower cervical, supraclavicular, and mediastinal lymph nodes. Most of the patients are adolescents or young adults, and the overall prognosis is excellent. It is characterized microscopically by the presence of a particular variant of the RS cell, the lacunar cell. This cell is large and has a single multilobated nucleus with multiple small nucleoli and an abundant, pale-staining cytoplasm. In formalin-fixed tissue, the cytoplasm often retracts, giving rise to the appearance of cells lying in empty spaces, or lacunae. The presence of collagen bands that divide the lymphoid tissue into circumscribed nodules. The cellular infiltrate may show varying proportions of lymphocytes, eosinophils, histiocytes, and lacunar cells. Classic RS cells are infrequent. Lacunar variants express CD15 & CD30. Mixed-Cellularity Hodgkin Lymphoma (MCHL): is the most common form of Hodgkin lymphoma in patients older than the age of 50 years with a male predominance. Classic RS cells are plentiful within a distinctive mixed cellular infiltrate, which includes small lymphocytes, eosinophils, plasma cells, and benign histiocytes. Compared with the other subtypes, patients with mixed cellularity have more disseminated disease and systemic manifestations. Lymphocyte-depleted Hodgkin disease (LDHL) (< 1% of cases): it is characterized by the presence of large numbers of RS that are often of bizarre morphology. Reactive cells are infrequent and fibrosis may be extensive. It is associated with older age and HIV positive status. Lymphocyte-rich HL (LRHL) (5% of cases): RS cells of the classic or la cunar type are observed with a background infiltrate of B-lymphocytes. Nodular lymphocyte predominant HL (NLPHL): NLPHL is a monoclonal B-cell tumor considered to be of germinal centre B- cell origin. It is characterized by formation of poorly defined nodules (nodular) or (nodular & diffuse) with proliferation of scattered large multilobated neoplastic cells LP (Lymphocyte- Predominant) cells in an inflammatory background, which contain a large number of small mature lymphocytes admixed with a variable number of benign histiocytes. Classic RS cells are difficult to find. Unlike classic RS cells, LP cells are: Positive for B-cell antigens (CD19, CD20, CD79a), Negative for CD15 & CD30. A diagnosis of NLPHD needs to be supported by immunohistochemical studies because it can appear similar to lymphocyte-rich HL or even some non-hodgkin lymphomas. The histologic diagnosis of Hodgkin lymphoma rests on the definitive identification of RS cells or their variants in the appropriate background of reactive cells. Immunophenotyping plays an important adjunct role in helping to distinguish Hodgkin lymphoma from reactive conditions and other forms of lymphoma. 5

Clinical Staging of Hodgkin (and Non-Hodgkin Lymphoma) (Ann Arbor Classification): Stage I: involvement of a single lymph node region (I) or involvement of a single extralymphatic organ or tissue (IE). Stage II: involvement of two or more lymph node regions on the same side of the diaphragm alone (II) or with involvement of limited contiguous extralymphatic organs or tissue (IIE). Stage III: involvement of lymph node regions on both sides of the diaphragm (III), which may include the spleen (IIIS), limited contiguous extralymphatic organ or site (IIIE), or both (IIIES). Stage IV: multiple or disseminated foci of involvement of one or more extralymphatic organs or tissues with or without lymphatic involvement. All stages are further divided on the basis of the absence (A) or presence (B) of the following systemic symptoms: significant fever, night sweats, unexplained loss of more than 10% of normal body weight. Etiology and Pathogenesis of HL: Hodgkin lymphoma (whether classical HL or NLPHL) is a neoplasm arising from germinal center B cells. All RS cell and its variants in Classical HL show somatic hypermutation that leads to Ig gene rearrangements and preclude expression of B cell markers, except in NLPHL variant (ongoing somatic hypermutation). Some cases are etiologically linked to EBV and incidence is increased in HIV-positive individuals. The EBV genome is present in the RS cells in up to 70% of cases of the mixedcellularity type and a smaller fraction of the nodular sclerosis type. Thus, EBV infection is likely to be a contributing step to the development of Hodgkin lymphoma, particularly the mixed-cellularity type. It has been found that the RS cells in classical forms of Hodgkin lymphoma, regardless of their EBV status, contain high levels of activated NF-κB, a transcription factor that normally stimulates B-cell proliferation and protects B cells from pro-apoptotic signals. Several EBV proteins that are known to activate NF-κB are expressed in EBV-positive RS cells. Thus, hyperactivation of NF-κB may be a central event in the genesis, growth, and survival of RS cells. The characteristic non-neoplastic, inflammatory-cell infiltrate seems to result from a number of cytokines secreted by RS cells, including IL-5 (which attracts and activates non-neoplastic, inflammatory cell infiltrate & eosinophils). These produce CD30 ligand which maintain the growth & survival of RS cells. Spleen: Splenomegaly: The spleen is frequently secondarily involved in a wide variety of systemic diseases. In virtually all instances, the response of the spleen causes its enlargement (splenomegaly). Causes of splenomegaly: Hematological: Chronic myeloid leukemia*. Chronic lymphocytic leukemia. Acute leukemia. Malignant lymphoma*. Primary myelofibrosis*. Polycythemia vera. Hairy cell leukemia. Thalassemia major or intermedia*. Sickle cell anemia (before splenic infarction). Hemolytic anemias. Megaloblastic anemia. 6

Portal hypertension: Cirrhosis. Hepatic. Portal. Splenic vein thrombosis. Storage diseases: Gaucher s disease*. Niemann-Pick disease. Histiocytosis X. Systemic diseases: Sarcoidosis. Amyloidosis. Collagen diseases: Systemic lupus erythematosus. Rheumatoid arthritis. Systemic mastocytosis. Infections: Acute: Septicaemia. Bacterial endocarditis. Typhoid. Infectious mononucleosis. Chronic: Tuberculosis. Brucellosis. Syphilis. Malaria. Leishmaniasis*. Schistosomiasis*. Tropical*: Possibly caused by malaria. * Possible causes of massive (>20 cm) splenomegaly. An enlarged spleen often removes excessive numbers of one or more of the formed elements of blood, resulting in anemia, leukopenia, or thrombocytopenia. This is referred to as hypersplenism, a state that can be associated with many of the diseases affecting the spleen listed above. In addition, platelets are particularly susceptible to sequestration in the red pulp; as a result, thrombocytopenia is more prevalent and severe in individuals with splenomegaly than are anemia or neutropenia. Thymus: Thymic Hyperplasia: Hyperplasia of the thymus is often associated with the appearance of lymphoid follicles, or germinal centers, within the medulla. These germinal centers contain reactive B cells, which are normally present in only low numbers in the thymus. Thymic follicular hyperplasia is present in most patients with myasthenia gravis and is sometimes also found in other autoimmune diseases, such as SLE and rheumatoid arthritis. Thymomas: These are tumors in which epithelial cells constitute the neoplastic element. Scant or abundant precursor T cells (thymocytes) are present in these tumors, but these are non-neoplastic. Thymomas are classified as follows Benign or encapsulated (60-70%): cytologically and biologically benign. Malignant thymoma: Type I (20-25%): cytologically benign but biologically aggressive and capable of local invasion and, rarely, distant spread. Type II (Thymic carcinoma) (5%): cytologically & biologically malignant. Gross features: Thymomas are lobulated, firm, gray-white masses up to 15 to 20 cm in longest dimension. Most appear encapsulated, but in 20% to 25% there is apparent penetration of the capsule and infiltration of perithymic tissues and structures. In thymic carcinomas metastases occur e.g. to the lungs. 7

Microscopic features: Almost all thymomas are made up of a mixture of epithelial cells and a variable infiltrate of non-neoplastic thymocytes (T-lymphocytes). Benign thymomas: show spindled or elongated epithelial cells that resemble those normally populate the medulla. In other tumors the above cells are admixed with plump, rounder, cortical-type epithelial cells; this pattern is referred to as a mixed thymoma. Malignant Thymoma Type I: is a tumor that is cytologically bland but locally invasive. They are composed of varying proportions of epithelial cells and reactive thymocytes; the epithelial cells have abundant cytoplasm and rounded vesicular nuclei. They often form palisades around blood vessels. Sometimes spindled epithelial cells are also present. The critical distinguishing feature is the penetration of the capsule and the invasion of surrounding structures. Malignant Thymoma Type II (Thymic Carcinoma): they are malignant cytologically. Most resemble either poorly or well-differentiated squamous cell carcinomas. The next most common malignant pattern is undifferentiated lymphoepithelioma-like carcinoma, which is composed of anaplastic cortical-type epithelial cells mixed with large numbers of benign thymocytes. 8