— SPECIALTY CONFERENCE —

Clinical History

A 69 year old man with a history of non-small cell lung carcinoma 8 years prior presents with a 2 month history of worsening shortness of breath, fatigue, weight loss, cough, and wheezing. He had been treated with both chemotherapy and radiation and continued to smoke 1/2 pack per day. A work-up revealed a large pleural effusion, plus intense right lung and pleural uptake on a PET scan. A pleural effusion was submitted to cytology for evaluation.

Pertinent Laboratory Data:

Pleural fluid WBC: 1069/µL; pleural fluid RBC: 3700/µL; pleural fluid protein: 4.6 g/dL Complete blood count showed Hematocrit 42%, WBC 14,000/µL, platelets 179,000/µL

Pathological/microscopic:

The ThinPrep showed mostly lymphoid cells with some showing irregular nuclei. Immunocytochemistry on the cell block showed most of the cells to be hematopoietic (LCA positive) with only rare cells expressing cytokeratin and calretinin consistent with mesothelial cells. Flow cytometry of fluid showed:

• Lymphoid cells: 90% of gated events
• T cells: 88%: normal CD4/CD8 ratio, no atypical immunophenotype
• B cells: 10%: lambda clonal population expressing CD45, CD19, CD20, CD5, CD38: all moderate-bright, negative for CD10, CD23, CD103, other markers

Patient continued to accumulate pleural fluid and had 3 taps with similar findings. A pleural catheter was placed and he drained a few hundred mL of fluid per day. He was admitted a month later for syncope and his WBC count was higher at 17,000. Flow cytometry showed the same monoclonal B cell population as noted in the pleural fluid and abnormal lymphocytes were noted in the blood. A FISH study of peripheral blood showed the CCND1 (11q23)- IgH (14q32) translocation. He was noted to have stable but widespread lymphadenopathy up to 1.7 cm on scans but no peripheral lymph nodes were palpated. Because of the history of lung cancer and the observation that only a minority of the pleural fluid lymphocytes were clonal, a pleural biopsy was performed to exclude two cancers. The pleural biopsy showed a predominance of B lymphocytes expressing CD20, PAX-5, CD5, and Cyclin D1. A Ki-67 stain showed 50% of the cells to be positive. CD3, CD10, and keratin stains were negative.

Differential Dx:

1. Lymphoma/lymphoproliferative disease

2. Lymphocyte rich reactive effusion

3. Carcinoma (neuroendocrine or poorly differentiated, history of lung carcinoma noted)

Final Dx:

Mantle cell lymphoma involving pleura, pleural fluid, and blood (presumed involvement of lymph nodes also)

Discussion:

Mantle cell lymphoma is an uncommon B cell lymphoma (3-10% of non-Hodgkin lymphoma). Mantle cell lymphoma is most common in older patient (median age 60-65) and there is a 2:1 male predominance. High stage disease is usually present including splenomegaly, blood and marrow involvement, and extensive lymphadenopathy. B symptoms are less common. Flow cytometry detects blood involvement in most cases, with 20-40% showing lymphocytosis. The GI tract may be involved including a lymphomatous polyposis picture in the large intestine. Other extranodal sites including skin, lung, salivary and CNS involvement are not uncommon, but CNS disease usually occurs late. A minority of cases (10-30%) present with indolent disease characterized by spleen and blood involvement (see WHO reference and clinical hematology references).

Cytology and histology usually shows monomorphic proliferation of small to medium sized lymphocytes with irregular nuclear shape. On histology, most cases are the classical variant with diffuse effacement of node architecture. Other variants include mantle zone pattern, small cell variant (resembling CLL/SLL), and a blastoid variant.

Ancillary studies and Genetics: Immunophenotyping most commonly shows: light chain restriction, CD20+, CD19+, CD5+, CD79b+, Cyclin D1+(nuclear staining on tissue), CD10-, CD23-, BCL2+, BCL6-. Molecular studies show a(11;14)(q13;32) in most cases, representing a juxtaposition of the CCND1 gene on chromosome 11 to the immunoglobulin heavy chain locus on chromosome 14, leading to the overexpression of cyclin D1 protein (encoded by CCND1 gene). Aberrant immunophenotypes can be seen, and some other lymphomas may show overlapping features if cyclin D1 or molecular studies are not incorporated. The presence of t(11;14) should be demonstrated if cyclin D1 is negative or equivocal, there is atypical immunophenotypes, morphology or clinical presentation. FISH can be performed on paraffin tissue or blood as well as cytology preparations. The SOX11 nuclear transcription factor has been recently described, and this is a helpful marker in the rare cases that are negative for cyclin D1 (these may have cyclin D2 or D3 expression and variant translocations). A high Ki-67 proliferation index (>40% correlates with 15 month median survival), p53 mutation, and p16 deletions are described in more aggressive variants including the blastoid type. Secondary molecular changes are relatively common.

Discussion of Differential Diagnosis: The differential diagnosis included a reactive lymphocytosis, metastatic malignancies, and other lymphoproliferative processes. A metastatic malignancy was unlikely as the cell population was entirely mesothelial and hematopoietic. No other cell types were seen in multiple taps. A benign lymphocyte predominant effusion can occur secondary to longstanding chronic effusion (congestive heart failure, etc), chronic infection (tuberculosis, fungal), autoimmune diseases,sarcoidosis, and other reactive causes. Chylothorax related to thoracic duct trauma will have a predominance of lymphocytes but also a chylous appearance. Reactive effusions tend to show a spectrum of lymphocytes (range of maturation) dominated by small lymphocytes but including reactive and transformed lymphocytes. Reactive effusions also commonly include plasmacytoid lymphocytes and plasma cells, so one should not jump to a diagnosis of myeloma when plasma cells are present. Lymphocytes may appear more atypical in effusions, both because of reactive and degenerative features (including irregular nuclei). Cytospin process may cause some distortion in cells and artifacts such as cell clustering. In reactive lymphocytosis, there is a predominance of T lymphocytes with normal immunophenotype. A predominance of B lymphocytes is suspicious for B cell lymphoma/lymphoproliferative process as most lymphocytes in reactive causes are T lymphocytes. A reactive lymphocytosis can also be associated with metastatic processes, including adenocarcinoma. Malignant causes of pleural effusion are more likely to result in patient symptoms including dyspnea, cough, and pain. Multiple taps may lead to a more diagnostic cytology specimen if metastatic process is suspected.

Lymphomas in pleural fluids will generally mimic the findings in fine needle aspiration, blood and tissue specimens. However, there may be some differences including more nuclear distortion because the cells are sitting in an effusion. Also, the preparation process may affect the cell appearance (cytospin artifact). Finally, in the case presented there were considerable numbers of intermixed reactive T cells. A combination of both Wright and Pap stains is useful, and one should always attempt to compare the cells to previous tissue, blood or marrow specimens for comparison.

In general, B-cell lymphomas tend to show a monomorphic proliferation of neoplastic cells in tissue and cytology samples. Chronic lymphocytic leukemia shows normal to slightly enlarged lymphocytes with round nuclei and often hyperclumped chromatin (cracked or "snickerdoodle" appearance). Only a few cells have nucleoli. Follicular lymphoma (low-grade) shows mostly small to medium lymphocytes with clefted nuclei. Follicular lymphoma can be very difficult to separate from mantle cell lymphoma, and ancillary studies are critical (immunophenotyping and/or molecular, see above). Marginal zone lymphoma (similar to MALT of lung) would be expected to show a spectrum of cells, predominantly small to medium round lymphocytes, and some with monocytoid or plasmacytoid appearance. There would be considerable overlap with reactive effusions, making immunophenotyping essential (this type of lymphoma is extremely rare in effusions).

Large cell lymphomas, primary effusion lymphomas, and blastic type lymphomas (Burkitt, lymphoblastic) were not in the differential diagnosis but are generally easier to diagnose as malignant on cytology. With high grade hematopoietic processes, one frequently observes high cell turnover in fluids, with apoptosis, mitoses, and necrotic cells. While lymphomas are stated to be monomorphic in overall appearance, there is frequently some variation in size of cells in effusions, both because of variable size of the lymphoma cells and the presence of intermixed benign cells. In general flow cytometry is preferable to immunocytochemistry for diagnosis of lymphoid effusions, as one can demonstrate light chain restriction and specific B-cell lymphoma immunophenotypes, and it is much easier to detect a small component of clonal cells within a background of T lymphocytes.

Review of Literature/Treatment:

Pleural involvement by systemic lymphoma is not uncommon (see series by Vega et al) but it is only rarely observed as a primary isolated lymphoma. The most common pleural lymphomas are large cell and follicular lymphoma, with mantle cell lymphoma reported. Czader and Ali have reported flow cytometry and cytology findings of mantle cell and other lymphomas in serous effusions. FISH for mantle cell lymphoma can be performed on cytospins of cytology specimens (see De Cunha Santos et al).

Pleural involvement by systemic lymphoma is not uncommon (see series by Vega et al) but it is only rarely observed as a primary isolated lymphoma. The most common pleural lymphomas are large cell and follicular lymphoma, with mantle cell lymphoma reported. Czader and Ali have reported flow cytometry and cytology findings of mantle cell and other lymphomas in serous effusions. FISH for mantle cell lymphoma can be performed on cytospins of cytology specimens (see De Cunha Santos et al).

The clinical hematology references included describe a mantle cell lymphoma international prognostic index (MIPI) that incorporates performance status, age, WBC count and LDH. Proliferative index is added if available. Median survival ranges from several years for low-risk group to 51 months for intermediate risk, and 29 months for high-risk groups. Standard chemotherapy does not generally result in long term remissions. Some indolent cases, especially in elderly patients, may be observed, and the rare localized cases can be treated with radiation. Older patients with intermediate or high risk MIPI have combination chemotherapy including Rituximab plus either Bendamustine (an alkylating agent) or CHOP. Younger symptomatic patients receive aggressive therapy including Rituximab plus HyperCVAD and high-dose cytarabine/methotrexate. Autologous stem cell transplantation is suggested for younger patients who achieve remission. For patients who relapse, newer therapies involve targeting known signaling pathways such as bortezomib (proteasome inhibitor) and immunomodulatory agents such as lenalidomide.The patient in this case was treated with a combination of Rituximab plus Bendamustine.

Conclusions:

1. Seek other information about your patients, including other laboratory and radiology data. When there is a question of lymphoproliferative process, check for abnormal hemogram/peripheral blood findings, don't rely on just anatomic pathology data.

2. Mantle cell lymphoma is generally aggressive, but some patients may pursue a more indolent course. Demonstration of molecular change (Cyclin D1 or CCND1 gene translocation) is recommended for definitive diagnosis.

3. Mantle cell lymphoma generally shows at least a mild degree of blood involvement. Blood is a simple source of material for ancillary studies when it is involved. Lymphomas in fluid may show considerable overlap with each other and with reactive effusions. Ancillary studies are almost always necessary when the lymphoma is composed of small to intermediate cells with nonblastic features.

4. Start using the NCCN guidelines to become familiar with what data is needed for basic patient management, and when additional ancillary tests might be indicated.

References

1. Czader M, Ali SZ. Flow cytometry as an adjunct to cytomorphologic analysis of serous effusions. Diagn Cytopathol 2003;29:74-78.
2. De Cunha Santos G, Ko HM, Geddie WR, et al. Targeted use of fluorescence in situ hybridization (FISH) in cytospin preparations. Cancer Cytopathol 2010;118:250-8.
3. DeMay RM. The Art & Science of Cytopathology. American Society for Clinical Pathology Press, 2012.
4. Jares P, Campo E. Advances in the understanding of mantle cell lymphoma. Br J Haematol 2008; 142:149-165.
5. McKay P, Leach M, Jackson R, et al. Guideline for the investigation and management of mantle cell lymphoma. Br J Haematol 2012; 159:405-26. PMID:22994971
6. Schwock J, Geddie WR. Diagnosis of B-cell non-Hodgkin lymphomas with small-/intermediate-sized cells in cytopathology. Patholog Res Int. 2012; 2012:164934. PMID:22693682
7. Vega F, Padula A, Valbuena JR, et al. Lymphoma involving the pleura: a clinicopathologic study of 34 cases diagnosed by pleural biopsy. Arch Pathol Lab Med2006;130:14970502.
8. Vose JM. Mantle cell lymphoma: 2012 update on diagnosis, risk-stratification, and clinical management. Am J Hematol 2012; 87:605-609.
9. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue / Edition 4. S Swerdlow, Editor. World Health Organization, 2008.
10. Zelenetz AD, et al. NCCN Clinical Practice Guidelines in Oncology: Non-Hodgkin's lymphomas, Version 3.2012. Available at www.nccn.org. Establish your own login (free to physicians).
11. Czader M, Ali SZ. Flow cytometry as an adjunct to cytomorphologic analysis of serous effusions. Diagn Cytopathol 2003;29:74-78.