Multiplex sequencing for EZH2, CD79B, and MYD88 mutations using archival cytospin preparations from B-cell non-Hodgkin lymphoma aspirates previously tested for MYC rearrangement and IGH/BCL2 translocation.

BACKGROUND: Gene rearrangements and specific translocations define some B-cell non-Hodgkin lymphoma (NHL) subtypes. Genome-wide mutational studies have revealed recurrent point mutations with prognostic implications. The goals of this study were to evaluate the feasibility of applying a multiplex mutation assay to archival cytospin preparations (CPs) and to investigate the rate of EZH2, CD79B, and MYD88 mutations in B-cell NHL samples previously tested for MYC rearrangement and/or IGH/BCL-2 translocation. METHODS: DNA was extracted from archival CPs of B-cell NHL cases with previous fluorescence in situ hybridization (FISH) assays for MYC rearrangement and/or IGH/BCL-2 translocation. Multiplex sequencing was performed for the detection of EZH2 (Y641), CD79B (Y196), and MYD88 (L265) mutations. Sanger sequencing was applied to samples with positive results and failed assays. RESULTS: Eighty-eight archival CPs were available from 40 patients. Alterations detected by FISH were: MYC rearrangement (10 cases), IGH/BCL-2 translocations (21 cases), dual translocations (6 cases), and other abnormalities for IGH/BCL-2 (23 cases) and for MYC (16 cases). DNA concentration ranged from 1.88 to 62.85 ng/µL (mean, 9.46 ng/µL). Successful results were obtained in 88.0% of the specimens submitted to multiplex sequencing. With Sanger sequencing, 2 additional mutated cases were found, and all cases with mutations were confirmed. Eight specimens showed mutations: 6 for EZH2, 1 for CD79B, and 1 for MYD88. Among them, 5 cases showed concurrent MYC and/or IGH/BCL-2 translocations and 2 revealed abnormal signals of IGH/BCL-2 and MYC. CONCLUSIONS: CPs archived for up to 6 years are a reliable source of high-quality genomic material for multiplex sequencing. Almost all B-cell NHL with point mutations showed concurrent chromosomal abnormalities.

MicroRNA signature obtained from the comparison of aggressive with indolent non-Hodgkin lymphomas: potential prognostic value in mantle-cell lymphoma.

PURPOSE: Mantle-cell lymphoma (MCL) has a variable natural history but is incurable with current therapies. MicroRNAs (miRs) are useful in prognostic assessment of cancer. We determined an miR signature defining aggressiveness in B-cell non-Hodgkin lymphomas (NHL) and assessed whether this signature aids in MCL prognosis. METHODS: We assessed miR expression in a training set of 43 NHL cases. The miR signature was validated in 44 additional cases and examined on a training set of 119 MCL cases from four institutions in Canada. miRs significantly associated with overall survival were examined in an independent cohort of 114 MCL cases to determine association with patient outcome. miR expression was combined with current clinical prognostic factors to develop an enhanced prognostic model in patients with MCL. RESULTS: Fourteen miRs were differentially expressed between aggressive and indolent NHL; 11 of 14 were validated in an independent set of NHL (excluding MCL). miR-127-3p and miR-615-3p were significantly associated with overall survival in the MCL training set. Their expression was validated in an independent MCL patient set. In comparison with Ki-67, expression of these miRs was more significantly associated with overall survival among patients with MCL. miR-127-3p was combined with Ki-67 to create a new prognostic model for MCL. A similar model was created with miR-615-3p and Mantle Cell Lymphoma International Prognostic Index scores. CONCLUSION: Eleven miRs are differentially expressed between aggressive and indolent NHL. Two novel miRs were associated with overall survival in MCL and were combined with clinical prognostic models to generate novel prognostic data for patients with MCL.

EZH2 and CD79B mutational status over time in B-cell non-Hodgkin lymphomas detected by high-throughput sequencing using minimal samples.

BACKGROUND: Numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHLs) have been revealed by novel high-throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex-associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B-cell NHLs, through use of a customized multiplex mutation assay. METHODS: DNA that was extracted from cytological material stored on FTA cards as well as from additional specimens, including archived frozen and formalin-fixed histological specimens, archived stained smears, and cytospin preparations, were submitted to a multiplex mutation assay specifically designed for the detection of point mutations involving EZH2 and CD79B, using MassARRAY spectrometry followed by Sanger sequencing. RESULTS: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y646) and CD79B (Y196) were detected in 13.2% and 8% of the samples, respectively, almost exclusively in follicular lymphomas and diffuse large B-cell lymphomas. In one-third of the positive cases, a wild type was detected in a different sample from the same patient during follow-up. CONCLUSIONS: Testing multiple minimal tissue samples using a high-throughput multiplex platform exponentially increases tissue availability for molecular analysis and might facilitate future studies of tumor progression and the related molecular events. Mutational status of EZH2 and CD79B may vary in B-cell NHL samples over time and support the concept that individualized therapy should be based on molecular findings at the time of treatment, rather than on results obtained from previous specimens. Cancer (Cancer Cytopathol) 2013;121:377-386. © 2013 American Cancer Society.

Negative images of crystalline immunoglobulin in crystal storing histiocytosis: A potential cytologic mimic of mycobacteria in smears.

Two cases are described of crystal storing histiocytosis (CSH) associated with extranodal marginal zone lymphoma, presenting as lung and subcutaneous masses respectively. Fine-needle aspiration of subcutis and smears prepared from the resected lung masses showed negative images. Cytology slides of both cases were reviewed to identify cytomorphological features for the differential diagnosis between immunoglobulin crystals and mycobacteria. The crystals in CSH consist of straight and needle shaped rods with pointed or angular edges and are more variable in thickness than the uniformly thin mycobacteria. Mycobacteria show a haphazard distribution, whereas crystals are frequently present in parallel arrays. Small lymphoid or plasma cells are identified in the background of CSH, whereas a necrotic and inflammatory background is seen in mycobacteriosis. Additional samples for culture in the case of mycobacteriosis, or flow cytometry and molecular clonality testing in the case of CSH can provide critical data for a definitive diagnosis.

Cytomorphologic findings of B-cell lymphomas with concurrent IGH/BCL2 and MYC rearrangements (dual-translocation lymphomas).

BACKGROUND: B-cell lymphomas with concurrent IGH/BCL2 and MYC gene rearrangements, termed dual-translocation or double-hit lymphomas (DTLs), rarely are identified. They usually are characterized by highly aggressive behavior, a poor prognosis, and complex karyotypes. The objective of this study was to review and describe the cytomorphologic findings in different types of cytologic preparations and clinicopathologic characteristics of patients with DTLs. METHODS: Patient samples with IGH/BCL2 and MYC rearrangements that were detected by fluorescence in situ hybridization during the period from October 2003 to September 2009 were selected for morphology review. Clinical data and results from additional studies were collected from patient reports. RESULTS: Cytologic samples from 14 patients (5 men and 9 women) were reviewed. The most common cytomorphologic pattern was a mixed cell population consisting predominantly of large cells (88.2%), mainly centroblasts (94.1%), with dark blue cytoplasm (76.4%) accompanied by apoptotic bodies (64.7%), with marked cellular pleomorphism (94.1%). Nuclear segmentation was present in 64.7% of samples, conferring a "coffee bean" nucleus, and cytoplasmic vacuoles were observed in 46.6% of samples. Immunophenotyping revealed the expression of CD20, CD19, surface immunoglobulin, and CD10 in 13 samples. Other chromosomal aberrations were also identified. Seven patients died of their disease, and the time from progression to death ranged from 1 month to 16 months. CONCLUSIONS: Large cells with deeply basophilic cytoplasm, cytoplasmic vacuoles, and frequent segmented nuclei, particularly in fine needle aspirate smears and especially in patients with clinically aggressive and/or unusual clinical features, should trigger a fluorescence in situ hybridization analysis for IGH/BCL2 and MYC translocation to identify this entity.

Whole stimulated salivary flow: correlation with the pathology of inflammation and damage in minor salivary gland biopsy specimens from patients with primary Sjögren's syndrome but not patients with sicca.

OBJECTIVE: To determine which measure of the salivary flow rate, stimulated or unstimulated, is most strongly associated with pathologic changes in minor salivary gland (MSG) biopsy specimens, and to explore the correlation of salivary flow with oral surface damage, disease duration, and symptom severity in patients with primary Sjögren's syndrome (SS). METHODS: In all patients (n = 32), a biopsy of the MSG was performed, and stimulated salivary flow was assessed. Beginning in 2002, unstimulated salivary flow was also assessed. Scores for the severity of symptoms, according to the decayed/missing/filled teeth (DMF) index, were recorded. Associations between measures of salivary flow and covariates characterizing pathology were examined. RESULTS: A definite association between stimulated salivary flow and the MSG focus score, the grade of MSG fibrosis, the duration of dry mouth symptoms, and the DMF score was observed. In contrast, unstimulated salivary flow was not associated with fibrosis, atrophy, the DMF score, or the duration of dry mouth symptoms. In patients with primary SS, the DMF score was associated with pathologic changes in the MSG. Among patients with sicca, 57.9% had an abnormal unstimulated salivary flow rate (versus 82.4% of patients with primary SS), and 15.2% had an abnormal stimulated salivary flow rate (versus 61.8% of patients with primary SS). Among patients with sicca, neither stimulated salivary flow nor unstimulated salivary flow was associated with the degree of fibrosis or atrophy or with the DMF score. CONCLUSION: Compared with unstimulated salivary flow, stimulated salivary flow appeared to be a better measure of inflammation (according to the focus score) and fibrosis. In patients with sicca, the unstimulated salivary flow rate appeared to be abnormal more commonly compared with the stimulated salivary flow rate. In the future, stimulated salivary flow may serve as a noninvasive surrogate biomarker of inflammation and fibrosis as well as a measure of response to treatment in patients with primary SS.

Effect of surgery to implant motion and force sensors on vertical ground reaction forces in the ovine model.

Activities of daily living (ADLs) generate complex, multidirectional forces in the anterior cruciate ligament (ACL). While calibration problems preclude direct measurement in patients, ACL forces can conceivably be measured in animals after technical challenges are overcome. For example, motion and force sensors can be implanted in the animal but investigators must determine the extent to which these sensors and surgery affect normal gait. Our objectives in this study were to determine (1) if surgically implanting knee motion sensors and an ACL force sensor significantly alter normal ovine gait and (2) how increasing gait speed and grade on a treadmill affect ovine gait before and after surgery. Ten skeletally mature, female sheep were used to test four hypotheses: (1) surgical implantation of sensors would significantly decrease average and peak vertical ground reaction forces (VGRFs) in the operated limb, (2) surgical implantation would significantly decrease single limb stance duration for the operated limb, (3) increasing treadmill speed would increase VGRFs pre- and post operatively, and (4) increasing treadmill grade would increase the hind limb VGRFs pre- and post operatively. An instrumented treadmill with two force plates was used to record fore and hind limb VGRFs during four combinations of two speeds (1.0 m/s and 1.3 m/s) and two grades (0 deg and 6 deg). Sensor implantation decreased average and peak VGRFs less than 10% and 20%, respectively, across all combinations of speed and grade. Sensor implantation significantly decreased the single limb stance duration in the operated hind limb during inclined walking at 1.3 m/s but had no effect on single limb stance duration in the operated limb during other activities. Increasing treadmill speed increased hind limb peak (but not average) VGRFs before surgery and peak VGRF only in the unoperated hind limb during level walking after surgery. Increasing treadmill grade (at 1 m/s) significantly increased hind limb average and peak VGRFs before surgery but increasing treadmill grade post op did not significantly affect any response measure. Since VGRF values exceeded 80% of presurgery levels, we conclude that animal gait post op is near normal. Thus, we can assume normal gait when conducting experiments following sensor implantation. Ultimately, we seek to measure ACL forces for ADLs to provide design criteria and evaluation benchmarks for traditional and tissue engineered ACL repairs and reconstructions.

Targeted use of fluorescence in situ hybridization (FISH) in cytospin preparations: results of 298 fine needle aspirates of B-cell non-Hodgkin lymphoma.

BACKGROUND: Fluorescence in situ hybridization (FISH) results from fine needle aspirates (FNA) of B-cell non-Hodgkin lymphomas (NHLs) were reviewed to 1) investigate the value added by using specific gene rearrangement probes to lymphoma diagnosis, prognosis, and subtyping; and 2) evaluate the prevalence of cytogenetic alterations other than specific translocations. METHODS: FISH results from assays performed on cytospin preparations from NHL FNAs over a 6-year period (2003-2009) were selected. Immunophenotyping, clinical data, and cytomorphologic data were reviewed according to the current World Health Organization (WHO) classification system. Hybridized probes, the purpose for the assay (subtyping or prognosis), and the cytogenetic abnormalities observed were retrieved from cytology reports. Data was categorized according to specific rearrangements and other chromosomal abnormalities. RESULTS: Successful results were obtained in 284 (95.3%) of 298 cases from 282 patients. Abnormalities were found in 216 (76%) cases and 68 (24%) did not show alteration. Among cases submitted for subtyping, 198 showed FISH-positive results, and specific gene rearrangements were found in 122 (61.6%) cases as follows: follicular 82, mantle cell 21, marginal zone 3, "dual hit" 13, and Burkitt lymphoma 3. In 21 cases, abnormalities were useful for prognosis. Nonspecific alterations alone or in combination with translocations were found in 98 cases. CONCLUSIONS: FISH performed on cytospin preparations was useful for confirmation of specific subclasses of NHL and may also provide valuable prognostic information. Cytogenetic abnormalities other than specific translocations were frequently found and could provide supportive evidence for a definitive diagnosis of lymphoma in FNA.

Two distinct syndromes of lymphoma-associated AL amyloidosis: a case series and review of the literature.

Light chain (AL) amyloidosis has a rare association with non-Hodgkin lymphoma (NHL). Both peritumoral and systemic AL amyloidosis have been reported, but a detailed description of these syndromes is lacking. We describe 10 patients with lymphoma associated AL amyloidosis. NHL patients with peritumoral amyloidosis had low or undetectable levels of monoclonal (M) protein, mostly single organ involvement(lung or soft tissue), and underlying extranodal marginal zone lymphoma, mucosa associated lymphoid tissue subtype. NHL patients with systemic amyloidosis had high levels of M-protein, multiorgan involvement with frequent cardiac involvement, and predominantly underlying lymphoplasmacytic lymphoma. Systemic amyloidosis was associated with inferior outcomes

Factor analysis of the clinical characteristics of primary Sjogren syndrome.

PURPOSE: The purpose of this study was to use factor analysis to analyze 90 clinical characteristics of a cohort of 231 patients with primary Sjogren syndrome (pSS). METHODS: The records of all patients seen at the University Health Network Sjogren Syndrome Clinic from October 1992 to July 2006 were reviewed and documented. Those diagnosed as pSS by the American European Consensus Criteria of 2002 were included. The 90 clinical variables, including health history, blood analysis, symptoms of dry eye and dry mouth, salivary flow and biopsy, tear flow and staining, were analyzed by factor analysis. RESULTS: Two hundred thirty-one patients with pSS charts were reviewed, and 90 variables were recorded. Factor analysis resulted in three factors: factor 1: ocular surface staining, factor 2: antimicrosomal antibodies and antithyroid antibodies, and factor 3: serum anti-Ro and anti-La. CONCLUSIONS: Ocular surface staining accounted for the greatest variance in this population of patients with pSS.

Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples.

BACKGROUND: MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue. RESULTS: Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p < 0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples. We also outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (30 < or = Ct < or = 35), and low (Ct>35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p < 0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance). CONCLUSION: Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

Classifying B-cell non-Hodgkin lymphoma by using MIB-1 proliferative index in fine-needle aspirates.

BACKGROUND: MIB-1 proliferation index (PI) has proven helpful for diagnosis and prognosis in non-Hodgkin lymphomas (NHLs). However, validated cutoff values for use in fine-needle aspiration (FNA) samples are not available. We investigated MIB-1 immunocytochemistry as an ancillary technique for stratifying NHL and attempted to establish PI cutpoints in cytologic samples. METHODS: B-cell NHL FNA cases with available cytospins (CS) MIB-1 immunocytochemistry results were included. Demographic, molecular, immunophenotyping and MIB-1 PI data were collected from cytologic reports. Cases were subtyped according to the current World Health Organization classification and separated into indolent, aggressive, and highly aggressive groups. Statistical analysis was performed with pairwise Wilcoxon rank sum test and linear discriminant analysis to suggest appropriate PI cutpoints. RESULTS: Ninety-one NHL cases were subdivided in 56 (61.5%) indolent, 30 (33%) aggressive, and 5 (5.5%) highly aggressive lymphomas. The 3 groups had significantly different MIB-1 PIs from each other. Cutpoints were established for separating indolent (<38%), aggressive (> or =38% to < or =80.1%) and highly aggressive (>80.1%). The groups were adequately predicted in 76 cases (83.5%) using the cutpoints and 15 cases showed discrepant PIs. CONCLUSIONS: MIB-1 immunohistochemistry on CS can help to stratify B-cell NHL and showed a significant increase in PI with tumor aggressiveness. Six misclassified cases had PIs close to the cutpoints. Discrepant MIB-1 PIs were related to dilution of positive cells by non-neoplastic lymphocytes and to the overlapping continuum of features between diffuse large B-cell lymphoma and Burkitt lymphoma. Validation of our approach in an unrelated, prospective dataset is required.

The role of fine-needle aspiration in the diagnosis of thyroid lymphoma: a retrospective study of nine cases and review of published series.

AIMS: To review the clinicopathological, cytomorphological and immunophenotyping data from new cases and published series of thyroid lymphoma diagnosed by fine-needle aspiration (FNA), in order to identify useful diagnostic features. METHODS: Cases from 1988 to 2009 with an FNA diagnosis of thyroid lymphoma were selected from hospital records. An electronic MEDLINE and EMBASE search retrieved published series from 1980 to 2009. Available clinical, cytomorphological and immunophenotyping data from all cases were collected. In our cases, cytology slides and available surgical specimens were also reviewed. RESULTS: There were nine cases from eight of our patients, and 70 reviewed cases from eight series with at least four patients each. The most common presentation was a rapidly enlarging thyroid mass. Average patient age was 61 years in reviewed cases and 72 years in our cases. Large-cell lymphoma was the predominant subtype, revealing relatively monotonous populations of large, abnormal lymphoid cells. One of our cases, later diagnosed as marginal zone lymphoma, showed small lymphocytes with plasmacytoid features. Immunoprofiling information was available in five of our cases (three by immunocytochemistry and two by laser scanning cytometry) and in 34 reviewed cases (22 by immunocytochemistry, six by flow cytometry, and six by flow cytometry or immunocytochemistry). CONCLUSIONS: Cytological diagnosis of thyroid lymphoma requires careful analysis of morphological, clinical and immunophenotypic information. The presented data suggest certain helpful features: a fast-growing nodule in an elderly patient, a monotonous population of large abnormal cells in a background of lymphoglandular bodies, a predominant population of plasmacytoid lymphocytes, and immunophenotyping demonstrating light chain restriction.

Rose bengal staining of the temporal conjunctiva differentiates Sjögren's syndrome from keratoconjunctivitis sicca.

PURPOSE: To compare the clinical presentation of 231 patients with primary Sjögren's syndrome (pSS) with 89 patients with aqueous-deficient dry eye (keratoconjunctivitis sicca; KCS), to determine those procedures that best differentiate these groups in the eye care clinic. METHODS: The records of all patients seen at the University Health Network Sjögren's Syndrome Clinic from October 1992 to July 2006 were reviewed and documented. The diagnosis of pSS was based on the AECC (American European Consensus Criteria) of 2002. KCS control subjects were non-SS patients with symptoms of dry eye and Schirmer scores of

Mantle cell lymphoma as a rare cause of intussusception: a report of 2 cases.

Intussusception is uncommon in adults and is only very rarely caused by malignant lymphoma. To our knowledge, there are only 2 previously reported cases of mantle cell lymphoma causing intussusception. We present 2 additional cases of intussusception at the ileocecal valve in patients being treated for mantle cell lymphoma, and a review of the pertinent literature is presented.

The reliability of lymphoma diagnosis in small tissue samples is heavily influenced by lymphoma subtype.

A specific pathologic diagnosis is important in malignant lymphoma because the diverse disease subtypes require tailored approaches to clinical management. Reliance on small samples obtained with cutting needles has been advocated as a less invasive alternative to using larger, excised samples. Although published studies have demonstrated the safety and apparent sufficiency of this approach in informing clinical care, none have systematically determined the accuracy of pathologic lymphoma subtyping based on very small samples. We used a tissue microarray representing 67 cases of malignant lymphoma and 17 samples of nonneoplastic lymphoid tissue to model lymphoma diagnosis in small samples. Overall, 73.8% of the cases were diagnosed with a level of confidence deemed sufficient for directing clinical management; 85.9% of these diagnoses were accurate. Small cell lymphomas with highly distinctive immunophenotypes, including small lymphocytic, mantle cell, and T-lymphoblastic lymphoma, were recognized most consistently and accurately in the small samples. In contrast, follicular lymphoma and marginal zone lymphoma were especially difficult. Our results indicate that the reliability of lymphoma diagnoses based on small samples is heavily influenced by lymphoma subtype.

CD30 and Epstein-Barr virus RNA expression in sclerosing angiomatoid nodular transformation of spleen.

Sclerosing angiomatoid nodular transformation (SANT) is a splenic lesion composed of angiomatoid/vascular nodules surrounded by hyalinized/sclerotic stroma, fibroblasts, myofibroblasts, and inflammatory cells. The endothelium within the nodules has a phenotype resembling splenic sinusoids, capillaries, and small veins. Martel et al. (Am J Surg Pathol 28:1268-1279, 2004) suggested that SANT may represent the final pathway of a variety of splenic lesions including inflammatory pseudotumors (IPTs). Epstein-Barr virus (EBV) has a role in the genesis of some splenic IPTs, but its presence in SANT has not been investigated. Six cases of SANT are reported. All were stained for CD31, CD34, CD8, CD68, smooth muscle actin, muscle-specific actin, and CD30 and were tested for EBV by in situ hybridization (EBER). All cases showed angiomatoid nodules with complex expression of CD31, CD34, and CD8, with focal CD68. Expression of CD30 by endothelial cells was also seen. One case had small diffuse areas lacking nodules resembling an IPT and was positive for EBV. The inflammatory cells and the normal spleen were negative for CD30 and EBER. In conclusion, SANT shows upregulation of CD30 with respect to normal spleen. The presence of EBV in the stromal cells of a case supports the notion that a subset of SANT may be related to IPT.

Lack of evidence of Epstein-Barr virus infection in patients with Castleman's disease. Molecular genetic analysis.

OBJECTIVE: Epstein-Barr virus (EBV) infection is associated with a diverse group of malignancies and many lymphoproliferative disorders. Castleman's disease (CD) is atypical lymphoproliferative disorder. The role of EBV in the pathogenesis of CD is not clear yet. The objective of this study is to investigate the EBV status in CD. METHODS: We searched medical records for cases of CD at the Toronto General Hospital, Toronto, Canada and King Abdulaziz University Hospital, Jeddah, Saudi Arabia. Twenty cases were found. The presence of EBV was analyzed using polymerase chain reaction. Polymerase chain reaction were performed at the Department of Pathology and Laboratory Medicine, Toronto General Hospital. The study started in 2001 and completed in 2005. RESULTS: The age range was 16-90 years. Seventeen patients manifested the localized form of CD. There were 11 males 9 females. Epstein-Barr virus genome was detected only in 2 cases; both were males and have plasma cell type. One is a localized type and the other is of a multicentric type. One patient revealed clonal rearrangement of the immunoglobulin H. CONCLUSION: The number of cases is small; however it appears that EBV is less likely to play a significant role in the pathogenesis of CD; however, it seems to be associated with clonal progression.