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

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1 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-hodgkin lymphomas. Cancer (Cancer Cytopathol) 2010;118: VC 2010 American Cancer Society. 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) ; szhan2@lsuhsc.edu Department of Pathology, Louisiana State University Health Science Center, Shreveport, Louisiana DOI: /cncy.20070, Received: September 30, 2009; Revised: November 18, 2009; Accepted: November 20, 2009, Published online March 25, 2010 in Wiley InterScience ( Cancer Cytopathology April 25,

2 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 medium. The specimens were processed using a routine laboratory protocol. Cell suspensions were stained with 4- fluorochrome-conjugated antibody combinations (fluorescein isothiocyanate, phycoerythrin, phycoerythrin- Texas 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; Beckman- Coulter). 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 T- cell 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). 106 Cancer Cytopathology April 25, 2010

3 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 Cases with FISH/PCR 1. FL Lymphoma Suggestive lymphoma PCR: IGH (þ); FISH: BCL2 (þ) 2. NK Lymphoma NK cell lymphoma PCR: Insufficient; FISH: MYC ( ) 3. FL Lymphoma Atypical PCR: IGH (þ), BCL2: (þ) 4. DLBL Atypical Negative PCR: IGH (þ); FISH: BCL6 (þ) Cases with FISH 5. FL Lymphoma FL FISH: BCL2 (þ) 6. FL Lymphoma FL FISH: MYC ( ) 7. B-cell lymphoma Lymphoma Atypical FISH: BCL2 ( ), BCL6 ( ) 8. FL Lymphoma Insufficient FISH: BCL2 (þ) 9. FL Lymphoma Lymphoma (CD10 ) FISH: BCL2 (þ) 10. B-cell lymphoma Lymphoma Lymphoma FISH: MYC ( ) 11. B-cell lymphoma Lymphoma Lymphoma FISH: MYC ( ), BCL6 ( ) Cases with PCR 12. Negative Atypical Atypical T cells PCR: TCR ( ) 13. B-cell lymphoma Suspicious for lymphoma Negative PCR: IGH (þ) 14. Negative Atypical Insufficient PCR: IGH ( ) 15. T-cell lymphoma Lymphoma Suspicious for T-cell lymphoma PCR: TCR-c (þ) 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, <10 4 ). PCR products were analyzed using conventional agarose gel electrophoresis. The PCR product sizes were from 80 base pairs (bp) to 300 bp (Mix 1b) and from 550 bp to 800 bp (Mix 1a) for the Mbr and from 600 bp to 1200 bp (Mix 2a) and from 500 bp to 1100 bp (Mix 2b) for the mcr. The internal amplification control was observed at 235 bp. Fluorescence In Situ Hybridization Study Cytospin slides for FISH analysis of chromosomal rearrangements t(14;18), t(11;14), and t(18;14) were prepared from cell suspensions of the FNA material. The slides were then fixed in 3:1 methanol-acetic acid followed by a 20-minute digestion in 10% pepsin/0.001 N HCl. The probes were then applied and denatured at 85 C for 1 minute and then incubated at 37 C for 16 to 18 hours. For BCL2, either a dual-color, break-apart rearrangement probe (targeting 18q21) or a dual-color, dual-fusion translocation probe [t(14;18)(q32;q21)] was used (Abbott Vysis, Inc., Abbott Park, Ill). After hybridization, the slides were washed to remove unbound probe (2 times standard saline citrate/npm40). The slides were counterstained using 4,6-diamidino-2-phenylindole and subsequently were viewed using a Leica DMR1 fluorescent microscope equipped with a Chroma 83,000 filter set and CytoVision software (Applied Imaging, Santa Clara, Calif). Before counting, the hybridization signals were screened for the presence of variant signal patterns. In each case, 100 to 200 nuclei were scored. The normal range for each probe was determined by using slides from patients who had no hematologic malignancies. Appropriate positive controls were selected from archival material of patients who had previous cytogenetic evidence of the reciprocal translocations. Representative images were captured using the CytoVision digitized system. RESULTS In total, 243 lymph node FNAs were performed during the study period. Ninety-six cases were positive, and 8 cases were atypical/suspicious for metastatic malignancies. The remaining 139 cases included 16 positive/suspicious for lymphoma, 15 atypical lymphoid proliferations, 73 Cancer Cytopathology April 25,

4 reactive, 14 granulomatous inflammation, and 21 nondiagnostic. In the 16 cases that were positive/suspicious for lymphoma, 13 cases had confirmatory flow cytometry, but 3 cases did not have adequate cells for flow cytometric analysis. Among the 15 atypical lymphoid proliferations, flow cytometry revealed that 1 was positive for lymphoma, 3 had atypical/suspicious flow cytometric results, 7 were negative, and 4 had insufficient material for flow cytometry. In total, 15 cases had molecular analysis performed on FNA materials. Four cases had both FISH and PCR tests, 7 cases had FISH tests only, and 4 cases had PCR tests only (Table 1). In the 4 cases with both PCR and FISH, 3 cases had either negative or atypical/suspicious flow cytometric results but a cytology diagnosis of malignant/suspicious for lymphoma (Fig. 1). PCR for IGH gene rearrangement was performed and confirmed the B- cell clonality in all 3 cases. FISH for BCL2 gene rearrangement was positive in 2 cases, and another case was positive for BCL6 gene rearrangement. One case of natural killer (NK) cell lymphoma was negative for myelocytomatosis viral oncogene homolog (MYC) gene rearrangement by FISH (for ruling out the possibility of Burkitt lymphoma) and had insufficient material to identify a TCR gene rearrangement using PCR. In the 7 FISH-only cases, 3 cases had positive BCL2 gene rearrangement (Fig. 2) for follicular lymphoma (flow cytometric results included 1 follicular lymphoma, 1 CD10-negative follicular lymphoma, and 1 case with insufficient cells). Three high-grade B-cell lymphomas were negative for MYC gene rearrangement, and 2 large B-cell lymphomas were negative for BCL6 gene rearrangement. In the 4 cases with PCR tests only, 1 B-cell lymphoma was positive for an IGH gene rearrangement, and 1 T-cell lymphoma was positive for a TCRG gene rearrangement. One reactive lymph node with atypical cytomorphology and an abnormal T-cell population on flow cytometry had negative PCR results for a TCR gene rearrangement, and another reactive lymph node with atypical cytomorphology and insufficient cells for flow cytometry had negative PCR results for IGH gene rearrangement. There were 45 cases with follow-up histologic examination (Table 2). Three false-negative cases included 1 Hodgkin lymphoma, 1 low-grade follicular lymphoma, and 1 large B-cell lymphoma. All 3 cases had negative B- cell clonality on flow cytometric analysis. Reviewing the cytologic slides, sampling error (no visible Reed-Sternberg cells on cytologic smears) accounted for the false-negative diagnosis of Hodgkin lymphoma. The case of low-grade Figure 1. A patient with diffuse large B-cell lymphoma had a cytologic diagnosis of suspicious for lymphoma but a negative flow cytometry result. Cytologic smear slides stained with (A) Diff-Quik and (B) Papanicolaou stain demonstrated hypocellular smears with some very large lymphocytes (original magnification, 400). Polymerase chain reaction analysis for immunoglobulin heavy-chain gene rearrangement was positive, and fluorescence in situ hybridization for B-cell chronic lymphocytic leukemia/lymphoma 6 (BCL6) gene rearrangement also was positive, supporting the diagnosis of diffuse large B-cell lymphoma. The follow-up excisional biopsy confirmed the cytologic diagnosis. follicular lymphoma was a challenging case because of the presence of a mixed population of lymphocytes with predominantly small lymphocytes and a negative flow cytometric result. One large B-cell lymphoma was misdiagnosed as granulomatous inflammation. Granulomatous inflammation was observed mixed with many large, atypical lymphocytes, but these atypical lymphocytes were overlooked by the cytopathologist. The negative flow cytometry analysis was a contributing factor for the false-negative diagnosis. We did not have any falsepositive cases during our study period. 108 Cancer Cytopathology April 25, 2010

5 FISH and PCR in Lymphoma FNA Diagnosis/Zhang et al Figure 2. A patient with of follicular lymphoma was positive for the t(14;18)(q32;q21) rearrangement. (A) A Vysis locusspecific identifier (LSI) B-cell chronic lymphocytic leukemia/ lymphoma 2 (BCL2) dual-color, break-apart probe for 18q21was used to detect rearrangements of the BCL2 gene region and was positive. The 2 abnormal cells in the field reveal 1 red signal, 1 green signal, and 1 fusion signal. (B) The Vysis LSI immunoglobulin heavy-chain (IGH)/BCL2 dual-color, dual fusion translocation probe was used to confirm the t(14;18)(q32;q21) translocation. The few abnormal cells in the field demonstrate 1 red signal (red labeling; 18q21), 1 green signal (green labeling; 14q32), and 1 orange signal (fusion signal for t[14;18]). DISCUSSION The debate about the role of FNA in the initial diagnosis of malignant lymphomas will continue, but using FNA biopsy for the diagnosis of lymphomas has become more popular in recent years. Several factors account for this change. One factor is the wide use of FNA biopsy for deeply located lymph nodes, including the use of conventional computed tomography (CT)/ultrasound (US)- guided FNA and EUS-FNA EUS-FNA allows more accurate sampling of smaller lymph nodes than CT/USguided FNA. 18 The minimally invasive nature of FNA biopsy is very attractive compared with other invasive modalities. The second factor that may account for the increased use of FNA biopsy for diagnosing lymphoma is the widely available multicolor flow cytometry, which can detect B-cell clonality and provides a very detailed immunophenotype of lymphoid neoplasms. Most B-cell lymphomas have characteristic immunophenotypic profiles, and they are very useful for lymphoma diagnosis and subclassification. 19 It has been documented that FNA in conjunction with flow cytometry for the diagnosis of hematopoietic processes has good sensitivity and specificity. For B-cell lymphomas, clonality can be confirmed by flow cytometric evidence of kappa or lambda light-chain restriction. Immunophenotypic profiles of the lesion can be helpful in the subclassification of a non-hodgkin B- cell lymphoma. Follicular lymphomas frequently are CD19-positive, CD10-positive, CD5-negative, and CD23-negative; small lymphocytic lymphomas/leukemias (SLL/CLL) characteristically are CD19-positive, CD10-negative, CD5-positive, and CD23-positive; mantle cell lymphomas often are CD19-positive, CD10-negatiave, CD5-positive, and CD23-negative; and marginal zone lymphomas frequently are CD19-positive, CD10- negative, CD5-negative, and CD23-negative. However, variation can be observed, such as CD10-negative; Table 2. Correlation Between Cytology and Histology Cytology, No. of Cases Histology Lymphoma ALP Negative Insufficient Total Lymphoma ALP Other malignancies 0 2 a Negative Insufficient Total ALP indicates atypical lymphoid proliferation. a One case was metastatic leiomyosarcoma, and another case was Kaposi sarcoma. Cancer Cytopathology April 25,

6 follicular lymphomas, CD23-negative SLL/CLL, and CD23-positive mantle cell lymphomas. Molecular tests for translocations and clonal gene rearrangement, such as t(14;18)(q32;q21) and BCL2 gene rearrangements, will be necessary in these variants. The third factor is the rapid expansion of molecular knowledge and diagnostic tests. Molecular tests have become a crucial component of modern pathology, especially in diagnosis of hematopoietic lesions. Genetic features play an important role in the classification of lymphoid malignancies, and many B-cell lymphomas have recurrent genetic alterations. 20 The PCR method for identifying clonality in FNAs is very useful in both B-cell and T-cell lymphomas. 21,22 Neoplasms of mature B cells usually represent a single clone of cells that expresses only 1 class of immunoglobulin light chain (kappa or lambda). The identification of a large, relatively pure population of light chain-restricted B cells is fairly straightforward using flow cytometric immunophenotyping and usually is reflected in an abnormal kappa-lambda ratio. 19 However, flow cytometric evaluation may fail to detect an abnormal population of B cells because of sampling error, cells loss during processing, a paucity of neoplastic cells, or difficult-to-identify cell populations. 19 When the cytomorphology is suspicious for lymphoma but flow cytometry results are inconclusive or negative, PCR analysis for IGH gene rearrangement or for immunoglobulin light-chain gene (IGK or IGL) rearrangements will be necessary to confirm the B-cell clonality. It has been demonstrated that PCR is very sensitive for identifying IGH gene rearrangement, and the high sensitivity (as few as 10 cells can produce a significant signal) make it suitable for detecting clonal populations when very low numbers of tumor cells are available. 11 However, both false-positive and false-negative results can occur, and correlation with clinical findings and cytomorphology is important during the interpretation of PCR results. In our study, 2 cases had a cytologic diagnosis of lymphoma but had atypical/suspicious flow cytometric results. Both cases were positive for clonal IGH gene rearrangements by PCR. Two cases with atypical/suspicious cytology but negative flow cytometric results also were positive for clonal IGH gene rearrangement by PCR. One case with suspicious cytology but insufficient material for flow cytometry was negative for clonal IGH gene rearrangement. In addition to detecting clonality, PCR also can be used to detect major and/or minor break points of BCL2 for the identification of t(14;18). Flow cytometric analysis may assist in the diagnosis and classification of mature T-cell and NK-cell lymphoid neoplasms; however, it is often more difficult to identify phenotypically abnormal T cells or NK cells than abnormal B cells. 19 TCR gene rearrangement analysis plays an important role in establishing T-cell clonality. We had several cases with an abnormal T-cell CD4/CD8 ratio, and a T-cell lymphoproliferative disorder was suspected. TCR gene rearrangement studies were negative for these cases, and they were signed out as reactive lymph nodes. One case of T- cell lymphoma had a positive TCRG gene rearrangement. However, pathologists should be cautious when using clonality studies to assign lineage, because clonal TCR gene rearrangement in B-cell malignancies and clonal immunoglobulin gene rearrangement in T-cell malignancies have been well documented. 21 FISH analysis is important to demonstrate the characteristic gene translocations for some lymphomas, and the specific translocations can be helpful for ruling in or out a specific diagnosis. 23,24 Most Burkitt lymphomas have MYC translocations at band 8q24 to the IGH (14q32) locus or, less commonly, to the IGL (22q11) or IGL (2p12) light-chain loci. Follicular lymphomas genetically are characterized by the translocation t(14;18)(q32; q21) and BCL2 gene rearrangement. The t(11;14)(q13; q32) between IGH and the cyclin D1 (CCND1) gene is present in almost all cases of mantle cell lymphoma and is considered the primary genetic event. Diffuse large B-cell lymphomas (DLBCLs) may have abnormalities of the 3q27 region involving the BCL6 gene in up to 30% of cases. Translocation of the BCL2 gene, a hallmark of follicular lymphoma, occurs in 20% to 30% of DLBCL, and an MYC gene rearrangement can be observed in up to 10% of DLBCL. Chromosomal translocations associated with extranodal marginal zone lymphoma of mucosaassociated lymphoid tissue (MALT lymphoma) include t(11;18)(q21;q21), t(1;14)(p22;q32), t(11;18)(q32;q21), and t(3;14)(p14.1;q32) and result in the production of a chimeric protein (apoptosis inhibitor 2 [API2]-MALT1) or in the transcriptional deregulation of the genes BCL10, MALT1, and forkhead box p1 (FOXP1), respectively. One patient in our study who had a previous history of follicular lymphoma had a CD10-negative immunophenotype according to flow cytometry, but cytomorphology was suggestive of recurrent follicular lymphoma. FISH analysis for this case was positive for t(14;18)(q32;q21) and confirmed the diagnosis of follicular lymphoma. The usefulness of FISH for detecting specific translocations in the diagnosis and classification of lymphomas on FNA has been reported in the literature. 1,12-14 In SLL/CLL 110 Cancer Cytopathology April 25, 2010

7 FISH and PCR in Lymphoma FNA Diagnosis/Zhang et al cases with a CD23-negative flow cytometry profile, the differential diagnosis includes mantle cell lymphoma and SLL/CLL. Demonstration of the t(11;14)(q13;q32) translocation by FISH between IGH and CCND1 will support the diagnosis of mantle cell lymphoma rather than SLL/CLL, because this translocation is present in almost all mantle cell lymphomas. The use of interphase FISH for the detection of t(11;14)(q13;q32) translocation in the diagnosis of mantle cell lymphoma on FNA specimens was discussed in a recent publication. 12 A breakapart probe can be used for screening purposes, and fusion probes can be used to detect the specific translocations if the screening test is positive. For example, a break-apart probe for 8q24 will detect all translocations that involve this region but cannot determine the specific translocation. By using fusion probes targeted to 8q24 and 14q32, or 22q11, or 2p12, FISH can detect the specific translocations for t(8;14)(q24;q32), t(8;22)(q24;q11), or t(2;8)(p12;q24). We propose using a systematic approach for handling lymph node FNA specimens (Fig. 3). Upon receiving slides for onsite evaluation, the first step is to determine the presence or absence of metastatic malignancies. If the cytology is positive for metastasis, then adequate materials should be obtained for a cell block. No flow cytometry should be submitted except for rare cases, such as the coexistence of metastasis and malignant lymphoma. If there is no evidence of metastatic malignancy, then all cases but suppurative lymphadenitis or necrotizing granulomatous inflammation should have materials submitted for flow cytometry. If Hodgkin lymphoma is strongly suspected after reviewing onsite smears, then using cell block materials may be more productive than flow cytometry. Some lymphomas may have a reactive cytomorphologic appearance, such as some low-grade lymphomas, T-cell lymphomas, and Hodgkin lymphoma; and granulomatous inflammation may be observed in Hodgkin lymphoma and in other malignant lymphomas. We recommend flow cytometric analysis for all cases except for the conditions mentioned above. If flow cytometric analysis is negative for B-cell clonality or has a normal T-cell population with a negative cytologic diagnosis, then the case can be signed out as negative for malignant lymphoma. If flow cytometry is inconclusive or if there is insufficient material with negative cytomorphology, then the case can be signed out as negative, but clinical correlation is recommended. If any clinical suspicion for malignant lymphomas is present, then PCR for clonality analysis (either B cells or T cells) should be performed. Figure 3. This is a proposed flow chart for handling fine-needle aspiration (FNA) lymph node specimens. IHC indicates immunohistochemistry; PCR, polymerase chain reaction; IGH, immunoglobulin heavy chain; IGK, immunoglobulin kappa light chain; IGL, immunoglobulin lambda light chain; TCR, T- cell receptor; FISH, fluorescence in situ hybridization. PCR for clonality analysis is recommended in cases with atypical flow cytometry results and/or an atypical/suspicious cytologic diagnosis, and FISH analysis for specific translocations usually follows or is performed simultaneously. In cases with positive flow cytometry and cytology results, FISH and PCR may be ordered for the subclassification of malignant lymphomas. In summary, FISH and PCR are important tools in lymphoma diagnosis and subclassification. They provide useful information for establishing T-cell and B-cell clonality and for the subclassification of malignant lymphoma by detecting a specific translocation. Molecular and cytogenetic studies play a significant role in resolving cases with either suspicious cytology or inconclusive flow cytometric analysis. Combining cytomorphology, flow Cancer Cytopathology April 25,

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