The role of fluorescence in situ hybridization and polymerase chain ...

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Original Article

The Role of Fluorescence In Situ Hybridization and Polymerase Chain Reaction in the Diagnosis and Classification of Lymphoproliferative Disorders on Fine-Needle Aspiration Songlin Zhang, MD, PhD, Fleurette Abreo, MD, Mary Lowery-Nordberg, PhD, Diana M. Veillon, MD; and James D. Cotelingam, MD

BACKGROUND: Fine-needle aspiration (FNA) has been used in the evaluation of lymphadenopathy for a long time and is highly reliable in the identification of metastatic malignancies. However, the role of FNA in the assessment of new lymphoproliferative disorders continues to be a subject of debate. The objective of the current study was to evaluate the role of molecular cytogenetic studies in FNA diagnoses of lymphoproliferative disorders. METHODS: A retrospective, computer-based search for lymph node FNAs from 2006 to 2007 was performed. Cases with either fluorescence in situ hybridization (FISH) and/or polymerase chain reaction (PCR) studies were subjected to further analysis. RESULTS: In total, 243 lymph node FNAs were performed during the period, including 104 that were positive/suspicious for metastatic malignancies, 16 that were positive/suspicious for lymphomas, 15 that demonstrated atypical lymphoid proliferation, 73 that were reactive, 14 that were deemed granulomas, and 21 that were determined to be nondiagnostic. Molecular analysis included combined FISH/PCR in 4 cases, FISH only in 7 cases, and PCR only in 4 cases. By using multiplex PCR, 6 cases with atypical/negative flow cytometry results were diagnosed as 4 B-cell lymphomas, 1 T-cell lymphoma, and 1 reactive lymph node; and 4 cases that had atypical T cells determined by flow cytometry were diagnosed as reactive. One CD10-negative follicular lymphoma and 2 cases with suspicious flow cytometry results were positive for t(14;18)(q32;q21) by FISH. Forty-five cases had follow-up histology with 3 falsenegative findings and no false-positive results. CONCLUSIONS: In this study, multiplex PCR studies for immunoglobulin heavy-chain or T-cell receptor gene rearrangements were useful for demonstrating clonality, and FISH studies were able to detect translocations or gene rearrangements that allowed for the subclassification of B-cell non-HodgC 2010 American Cancer Society. kin lymphomas. Cancer (Cancer Cytopathol) 2010;118:105–12. V KEYWORDS: fluorescence in situ hybridization, polymerase chain reaction, fine-needle aspiration, lymphoma, lymph node.

The role of fine-needle aspiration (FNA) biopsy on initial evaluation of lymphoproliferative disorders has been under

intensive debate for a long time, but the value of FNA biopsy on recurrent lymphoma has been established.1-3 The argument against the use of FNA on initial lymphoma diagnosis is that FNA specimens lack histologic patterns. However, the increasing role of immunophenotype and molecular cytogenetics in the diagnosis and classification of non-Hodgkin lymphomas may dramatically change opinions about using FNA biopsy for the evaluation of lymphoproliferative disorders. Currently, FNA combined with flow cytometry is used increasingly for the evaluation of both new and recurrent lymphomas.3-9 FNA biopsy often provides enough materials for flow cytometric analysis and molecular cytogenetic tests. Molecular tests, such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR), have been used on FNA specimens in recent years.1,10-14 The combination of cytomorphology and ancillary studies (including flow cytometry and molecular tests) can classify most B-cell non-Hodgkin lymphomas. An open biopsy from a superficial, enlarged lymph Corresponding author: Songlin Zhang, MD, PhD, Department of Pathology, Louisiana State University Health Science Center, 1501 Kings Highway, PO Box 33932, Shreveport, LA 71130; Fax: (318) 675-4568; [email protected] Department of Pathology, Louisiana State University Health Science Center, Shreveport, Louisiana DOI: 10.1002/cncy.20070, Received: September 30, 2009; Revised: November 18, 2009; Accepted: November 20, 2009, Published online March 25, 2010 in Wiley InterScience (www.interscience.wiley.com)

Cancer Cytopathology

April 25, 2010

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Original Article

node may be easy, but open biopsy for a deeply located lymph node is much more invasive than FNA biopsy. In recent years, the introduction of endoscopic ultrasound (EUS)-guided FNA (EUS-FNA) has made FNA a more convenient way to sample a deeply located lymph node.15 Many clinical scenarios for the FNA diagnosis of lymphoma are from patients with lymphadenopathy and were undertaken to rule out metastatic malignancy during the patient’s initial evaluation. So, the importance of cytopathologists having good cytomorphologic skills and being familiar with the available ancillary tools for lymphoma diagnosis cannot be overemphasized. At the Louisiana State University Health Science Center-Shreveport, we have very sophisticated flow cytometry and molecular genetic laboratories. Cytopathologists provide onsite evaluation and triage of all FNA biopsy specimens. Herein, we share our experience of the past 2 years.

MATERIALS AND METHODS Data Collection A retrospective search for lymph node FNA biopsies obtained between January 1, 2006 and December 31, 2007 was performed using the database of the Department of Pathology, Louisiana State University Health Science Center-Shreveport. Then, cross searches for corresponding ancillary studies (flow cytometry and molecular genetic tests) and follow-up histology were performed. False-positive and false-negative diagnoses were determined based on the histologic diagnosis. Cytologic Examination FNA biopsies were performed by pathologists, interventional radiologists, or clinicians. Conventional mirror slides were prepared on each pass. Diff-Quik stain was used for onsite evaluation on half of the prepared mirror slides, and Papanicolaou stain was used for the remaining half of the prepared slides. If the onsite rapid interpretation was negative for metastatic malignancy, then additional passes were performed on all cases for flow cytometric analysis except in cases of purulent lymphadenitis or necrotizing granulomatous inflammation. FNA materials were placed in RPMI-1640 cell culture medium and were submitted for flow cytometric analysis. Flow Cytometry Flow cytometric immunophenotyping was performed on FNA specimens collected in RPMI-1640 cell culture me-

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dium. The specimens were processed using a routine laboratory protocol. Cell suspensions were stained with 4fluorochrome-conjugated antibody combinations (fluorescein isothiocyanate, phycoerythrin, phycoerythrinTexas Red, and R-phycoerythrin-cyanin 5) according to the limited lymphoma panel routinely used on FNA specimens or on other specimens with limited cellularity. The antibodies used in the panel included antibodies against the following cluster of differentiation (CD) antigens: CD45, CD3, CD4, CD5, CD8, CD10, CD19, CD20, CD23, FMC-7, kappa, and lambda (Beckman Coulter, Miami, Fla and Dako Cytomation, Carpinteria, Calif). Approximately 5000 gated events per tube were acquired on the flow cytometer (FC500; BeckmanCoulter). The results were analyzed using the CXP computer software program (Beckman-Coulter). Polymerase Chain Reaction Cells for molecular studies were prepared by centrifugation and pellet retrieval followed by resuspension in a buffered solution that was designed to lyse cell membranes while preserving nucleic acids. Genomic DNA was prepared from each sample using standard laboratory protocols for nucleic acid extraction (QIAamp DNA Mini Kit; Qiagen, Inc., Valencia, Calif). After extraction, DNA was rehydrated and then quantified using traditional spectrophotometry (A260/A280 ratio). Immunoglobulin heavy-chain (IGH), TCR beta (TCRB), and TCR gamma (TCRG) gene rearrangements were evaluated using a multiplex PCR assay (Invivoscribe, San Diego, Calif). Briefly, for immunoglobulin gene rearrangement assay kit framework regions, Standardized Master Mixes 2 and 3 were used for multiplex PCR analysis of B-cell clonality, and Master Mix 1 was used as the amplification control. The TCR assay kit amplifies regions of patient DNA to assess the presence of clonal Tcell populations. PCR products were analyzed on an ABI310 gene analyzer (Applied Biosystems, Inc., Foster City, Calif). Clonal B-cell or T-cell populations were determined by the detection of 1 or 2 peaks within the defined regions of the PCR targets. Results for B-cell and T-cell assays were reported as clonal, oligoclonal, polyclonal, or indeterminate for clonality according to standard laboratory protocols. Appropriate positive and negative controls were used. PCR assays for the detection of B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2 (BCL2) gene rearrangements were performed according to standard laboratory protocol using a commercial kit (Invivoscribe).

Cancer Cytopathology

April 25, 2010

FISH and PCR in Lymphoma FNA Diagnosis/Zhang et al

Table 1. Final Diagnoses, Cytologic Diagnoses and Flow Cytometry and Fluorescence In Situ Hybridization/Polymerase Chain Reaction Test Results

Case/Diagnosis

Cytology Diagnosis

Flow Cytometry Results

FISH or PCR Results

Lymphoma Lymphoma Lymphoma Atypical

Suggestive lymphoma NK cell lymphoma Atypical Negative

PCR: PCR: PCR: PCR:

IGH (þ); FISH: BCL2 (þ) Insufficient; FISH: MYC () IGH (þ), BCL2: (þ) IGH (þ); FISH: BCL6 (þ)

Lymphoma Lymphoma Lymphoma Lymphoma Lymphoma Lymphoma Lymphoma

FL FL Atypical Insufficient Lymphoma (CD10) Lymphoma Lymphoma

FISH: FISH: FISH: FISH: FISH: FISH: FISH:

BCL2 (þ) MYC () BCL2 (), BCL6 () BCL2 (þ) BCL2 (þ) MYC () MYC (), BCL6 ()

Atypical Suspicious for lymphoma Atypical Lymphoma

Atypical T cells Negative Insufficient Suspicious for T-cell lymphoma

PCR: PCR: PCR: PCR:

TCR () IGH (þ) IGH () TCR-c (þ)

Cases with FISH/PCR 1. 2. 3. 4.

FL NK FL DLBL

Cases with FISH 5. FL 6. FL 7. B-cell lymphoma 8. FL 9. FL 10. B-cell lymphoma 11. B-cell lymphoma

Cases with PCR 12. 13. 14. 15.

Negative B-cell lymphoma Negative T-cell lymphoma

FISH indicates fluorescence in situ hybridization; PCR, polymerase chain reaction; FL, follicular lymphoma, IGH, immunoglobulin heavy-chain rearrangement; BCL2, B-cell chronic lymphocytic leukemia/lymphoma 2; (þ), positive; NK, natural killer cell lymphoma; (), negative; DLBL, diffuse large B-cell lymphoma; TCR, T-cell receptor rearrangement; TCR-c, T-cell receptor gamma chain.

Briefly, BCL2 gene rearrangement t(14;18)(q32;q21) was detected using a multiplex PCR analysis with primers to target the joining region of the IGH region (Mbr) or the minor cluster region (mcr). The remaining primer cocktail targets a ubiquitous human leukemic antigen (HLA) class II gene to ensure that sufficient DNA quality and quantity are present to generate a valid result. The limit of detection using nested amplification is less than 1 BCL2 translocation-positive cell in 10,000 normal cells (limit of detection,