—  SPECIALTY CONFERENCE  —

Cytopathology

Case 7 - B-Cell Lymphoma

Christina Kong
Stanford University School of Medicine
Stanford, CA


Click on each slide thumbnail image for an enlarged view
Clinical History
A 44-year-old man presents with a 2 cm right inguinal nodule, which has been present for 4-5 years and is stable in size. Four months prior, he underwent FNA biopsy of a left parotid nodule at an outside institution; cytology and flow cytometry were consistent with a benign lymph node. An FNA biopsy of the right inguinal nodule is performed (Figures A,B,C).


Case 7 - Figure A - Cellular smear shows variably sized but predominantly small lymphoid cells that appear cytologically unremarkable. A follicular dendritic cell is present in the field for size comparison (Papanicolaou, 400X).
Case 7 - Figure B - Predominantly small lymphoid cells with intermixed larger cells and a tingible-body macrophage (Diff-Quik, 400X).
Case 7 - Figure C - Higher power view of lymphoid cells. Note the presence of occasional clefted and angulated lymphoid cells, as well as numerous lymphoglandular bodies in the background (Diff-Quik, 600X).

Cytologic Findings
Aspirate smears are cellular and show a mixed lymphoid population composed of predominantly small lymphocytes and scattered intermediate sized forms. The lymphocytes exhibit irregular nuclear contours. Rare tingible body macrophages are present. No significant large cell component is identified.

Flow Cytometric Findings
Flow immunophenotyping shows a lambda monotypic B-cell population, which expresses CD19, CD20, CD10 and CD23. There is no CD19/CD5 co-expression.

Immunophenotypes of Small B-cell Lymphomas

  CD5 CD23 CD10
SLL/CLL + + -
Mantle Cell + - -
Marginal Zone - - -
Follicular - +/- +

Histology
The patient underwent excision of his left parotid nodule at an outside institution prior to the right inguinal FNA. Histologic sections showed an intraparenchymal lymph node effaced by follicles composed of small cells. No lymphoepithelial lesions or tingible body macrophages were identified. Immunoperoxidase stain for CD20 showed a predominance of B-cells, suggestive of lymphoma. However, the stain for bcl-2 stain (positive in follicular lymphomas – 100% grade 1, 85% grade 2, 75% grade 3) was equivocal. [1] Molecular studies were performed to confirm the diagnosis of B-cell lymphoma. These studies, which were pending at the time of the right inguinal FNA, showed a clonal immunoglobulin gene rearrangement but no evidence of a t(14;18) translocation.

Molecular Studies

Detection of B-cell clonality by PCR
Immunoglobulin heavy chain (IgH) gene rearrangements occur early in B-cell development. Since they are unique to each cell, these gene rearrangements can be used as a marker of clonality.

Identification of a clonal population should not be interpreted as diagnostic of malignancy. Non-malignant clonal populations can be seen transiently in the setting of infection or autoimmune disorders. [2, 3, 4, 5] False positive results have also been reported when PCR is performed on small or microdissected samples. [6] In addition, false negative results can occur due to sampling error, sample handling (PCR assay inhibited by heparin), or minimal detection limits. The results of this test need to be interpreted in the context of pathologic and clinical findings.

VDJ (FR3) Assay:

- Uses VH-FR3 and JH primer set

- Detection limit: 10-15% clonal cells in a polyclonal background

- If negative, FR1c assay is performed

FR1c Assay:

- Uses VH-FR1c primers

- Detection limit: 5% clonal cells in a polyclonal background (Stanford)

- Decreased specificity: Higher detection of non-malignant clonal populations

The patient's sample (Pt) showed the presence of a clonal IgH gene rearrangement:


Pt(+)(+)(-)

Detection of t(14;18) by nested PCR
The t(14;18)(q32;q21) translocation is present in 80-85% of follicular lymphomas. [1, 7] It occurs when the bcl-2 gene on chromosome 18 is moved to one of the immunoglobulin heavy chain (JH) regions on chromosome 14. The translocation brings the bcl-2 gene into the domain of the IgH enhancer, leading to overexpression of the bcl-2 protein. Since bcl-2 blocks apoptosis (programmed cell death), overexpression leads to decreased cell turnover. [1] This can result in the accumulation of a monoclonal cell population and the development of lymphoma.

However, detection of a t(14;18) translocation is not diagnostic of follicular lymphoma. Rare cells with t(14;18) have been found by PCR in reactive lymphoid tissue from normal individuals. [8, 9] This suggests that bcl-2 translocations alone are not sufficient for the development of follicular lymphoma.

False negative results can be due to sampling error, sample handling (PCR assay inhibited by heparin), or minimal detection limits. In addition, not all t(14;18) breakpoints are detected. [1] The current PCR assay uses primers for two breakpoint cluster regions – major breakpoint region (MBR) and minor cluster region (mcr). If the translocation is outside of these two regions, it will not be detected. The false negative rate is approximately 5-10%.

The patient's sample (Pt) did not show the presence of a t(14;18) translocation:

PtPtMBRmcr(-)

Discussion: Low Grade Follicular Lymphoma
FNA biopsy with flow cytometry is a rapid, cost effective, minimally invasive method for evaluating lymph nodes, both in patients with new onset lymphadenopathy and in those with a history of lymphoma.

The World Health Organization (WHO) lymphoma classification system relies less on architecture and allows for the definitive subclassification of many lymphomas based on the cytologic appearance of the lymphoid cells and the immunophenotype. [10] Subsequently, flow cytometry plays an essential role in the cytologic diagnosis of lymphoma. [11, 12, 13, 14] It not only allows for the subclassification of lymphomas based on the immunophenotype but can also detect the presence of a small monoclonal population in a background of polyclonal lymphoid cells. Flow cytometry is especially important in the diagnosis of low grade lymphomas since they can appear cytologically identical to a reactive lymph node, including the presence of scattered tingible body macrophages. The differential diagnosis for a reactive cytologic appearance includes reactive hyperplasia, low grade lymphoma, partial involvement by lymphoma and Hodgkin lymphoma. While flow cytometry can distinguish between the first three entities, it is not helpful in the diagnosis of Hodgkin lymphoma. If a diagnosis of Hodgkin lymphoma is suspected, it is best to obtain material for immunohistochemical stains.

Cytologic material can also be submitted for cytogenetic (e.g. FISH for c-myc) or molecular diagnostic studies (e.g. PCR for B-cell clonality). Cytogenetic studies require fresh material but molecular studies can be performed on fixed material. Given the possibility of false positive results (see "Molecular Studies" section), these studies should be interpreted in the context of cytologic, flow immunophenotypic and clinical findings.

In this case, the diagnosis of low grade follicular lymphoma was based on the presence of a predominantly small lymphoid population and the flow cytometry findings of a CD10(+) lambda restricted population of B-cells. The presence of CD10 expression is characteristic of follicular lymphomas but is not seen in all cases. It is present in 60% of cases by immuohistochemistry and 90% of cases by flow cytometry. [11] However, it should also be kept in mind that CD10 expression can be seen with Burkitt's lymphoma, some diffuse large B-cell lymphomas, precursor B- or T-lymphoblastic lymphoma/leukemia, and normal cortical thymocytes. Therefore it is important to correlate the presence of CD10 expression with the cytologic appearance of the lymphoid cells and the clinical presentation.

By the WHO Classification, follicular lymphomas are divided into three grades based on the proportion of small and large cells – grade 1, predominantly small cleaved; grade 2, mixed small cleaved and large cell; grade 3, follicular large cell. However, only two grades are clinically significant. Low-grade (grades 1 and 2) follicular lymphomas are indolent while high-grade (grade 3) follicular lymphomas are aggressive and require treatment. Based on aspirate smears, it is often possible to distinguish between a low-grade and high-grade follicular lymphoma but, as in tissue, there is less reproducibility with the use of three grades. [15] Two different methods for grading follicular lymphomas are generally used but there are no well-established criteria for grading based on cytologic material. The two methods parallel those used in tissue sections – visual estimation of the percentage of large cells or a count of transformed lymphocytes. [1, 16] At Stanford, the visual estimation method is used. In cases where the cytologic appearance is not clearly consistent with a low grade follicular lymphoma or where the clinical presentation is discordant with the cytologic findings, an excisional biopsy is recommended for further evaluation.

In patients with a history of lymphoma, FNA biopsy is often used to evaluate for transformation of a low grade lymphoma to a high grade lymphoma. In cases where the aspirate smears show predominantly small cells or sheets of large cells, the diagnosis is clear-cut. However, for the in-between cases, there are not well-established criteria for diagnosing transformation based on a cytologic specimen. Various studies have looked at the utility of proliferation markers (PCNA or Ki-67), transformed lymphocyte count and analysis of cell size by flow cytometry in the diagnosis of transformation. [16, 17, 18] These methods appear to show some correlation with grade but more studies are needed.

Triaging specimens for evaluation of lymphoid lesions:

For each aspirate, place a small drop on a slide and make two smears (one air-dried, one fixed).
Rinse the remainder of the specimen in a small amount (~5 mL) of RPMI (holding media).
Repeat aspirations until RPMI appears cloudy and small particles are seen when the fluid is agitated. This can be obtained with a minimum of two to three passes but, depending on technique, may require more.
Perform immediate evaluation of aspirate smear to confirm lymphoid nature of specimen and send specimen for flow cytometry.
If background is suggestive of Hodgkin lymphoma, use material in RPMI for immunohistochemical stains.
If specimen is insufficient for a full panel, perform limited flow cytometry panel (e.g. two tubes – CD19/CD5, kappa/lambda – can diagnose B-cell lymphomas by the presence of light chain restriction and CD19 expression; CD5 expression can allow further sub-classification)
If possible, obtain additional material for cell block, in case the flow cytometry results indicate a non-hematolymphoid process or are inconclusive.

References

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  2. Calvert RJ, Evans PA, Randerson JA, Jack AS, Morgan GJ, Dixon MF. The significance of B-cell clonality in gastric lymphoid infiltrates. J Pathol. Sep 1996;180(1):26-32.
  3. Ferraccioli GF, Sorrentino D, De Vita S, et al. B cell clonality in gastric lymphoid tissues of patients with Sjogren's syndrome. Ann Rheum Dis. May 1996;55(5):311-316.
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  7. Nathwani B, Harris N, Weisenburger D, et al. Follicular lymphoma. In: Jaffe E, Harris N, Stein H, Vardiman J, eds. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2001:162-167.
  8. Limpens J, de Jong D, van Krieken JH, et al. Bcl-2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia. Oncogene. Dec 1991;6(12):2271-2276.
  9. Aster JC, Kobayashi Y, Shiota M, Mori S, Sklar J. Detection of the t(14;18) at similar frequencies in hyperplastic lymphoid tissues from American and Japanese patients. Am J Pathol. Aug 1992;141(2):291-299.
  10. Harris NL, Jaffe ES, Diebold J, et al. World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting-Airlie House, Virginia, November 1997. J Clin Oncol. Dec 1999;17(12):3835-3849.
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  15. Young NA, Al-Saleem T. Diagnosis of lymphoma by fine-needle aspiration cytology using the revised European-American classification of lymphoid neoplasms. Cancer. Dec 25 1999;87(6):325-345.
  16. Young NA, Al-Saleem TI, Al-Saleem Z, Ehya H, Smith MR. The value of transformed lymphocyte count in subclassification of non-Hodgkin's lymphoma by fine-needle aspiration. Am J Clin Pathol. Aug 1997;108(2):143-151.
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