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CASE HISTORY

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED
  7. Supporting Information

The patient is a 22-year-old Caucasian male presenting with chest tightness, cough, and dyspnea on exertion. He first noticed these symptoms 3 months prior to his current admission. A chest CT showed a large mediastinal mass (15 cm × 11 cm × 7 cm) with areas of central necrosis encasing the major central vessels and airways. In addition, he was found to have bulky supraclavicular, internal mammary, and cardiophrenic adenopathy. He subsequently underwent a supraclavicular lymph node excisional biopsy.

FLOW CYTOMETRIC STUDIES

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED
  7. Supporting Information

The tissue specimen was immunophenotyped with the following panel of antibodies, listed as fluorescein isothiocyanate/phycoerythrin/peridinin chlorophyll protein/allophycocyanin combinations: G2a/G1/G2a/G1 (isotype control tube), CD10/CD19/CD20/CD38, CD30/CD2/CD45/CD3, polyclonal kappa/polyclonal lambda/CD5/CD19, monoclonal kappa/monoclonal lambda/CD20/CD38, CD7/CD4/CD8/CD3, and FMC-7/CD23/CD5/CD19. CD2 (55.2), CD3 (SK7), CD4 (SK3), CD5 (L17F12), CD7 (4H9), CD8 (SK1), CD10 (W8E7), CD19 (SJ25C1), CD20 (L27), CD23 (EBV-CS5), CD38 (HB7), CD45 (2D1), FMC-7 (FMC7), monoclonal immunoglobulin kappa (TB28-2), and monoclonal immunoglobulin lambda (I-155-2) antibodies were obtained from Becton Dickinson (San Jose, CA). Antibodies to polyclonal immunoglobulin kappa (goat) and lambda (goat) were obtained from Coulter-Immunotech (Hialeah, FL). Anti-CD30 (Ber-H2) was obtained from DAKO (Carpinteria, CA). At least 75,000 events were acquired using a FACSCanto flow cytometer with FACSDiva software (Becton Dickinson, San Jose, CA). Data analysis was performed using Paint-A-Gate software (Becton Dickinson). Positivity for an antigen was defined as at least 20% of lymphoma events beyond a 2% isotype control threshold. List mode files for the following tubes are available for download from the journal website (see Supporting Information): Tube 3 (CD30/CD2/CD45/CD3) and Tube 6 (CD7/CD4/CD8/CD3) along with images of example analysis.

DISCUSSION

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED
  7. Supporting Information

Immunophenotypic analysis revealed a 75–78% population of variably sized cells that were CD2(+), CD3(+), CD4(+), CD5(+), CD7(+), CD8(−), CD10(−), CD19(−), CD20(−), CD23(−), CD30 (partial +), CD38 (variably +), CD45 (bright +), and FMC-7(−) (Fig. 1; see Supporting Information). These findings, correlated with the presence of large, atypical cells in the flow cytometry cytospin preparation (Fig. 2) suggested a differential diagnosis including an anaplastic large cell lymphoma, ALK(+) or ALK(−), and a peripheral T-cell lymphoma, not otherwise specified. However, histologic examination of the lymph node sections showed diffuse architectural effacement by a cellular process with nodular distribution, and focal thickening of the lymph node capsule and thick bands of fibrosis throughout the tissue. The mixed cellular infiltrate consisted of small lymphocytes, occasional plasma cells, and rare neutrophils and eosinophils, admixed with numerous clusters of medium to large atypical cells with irregular nuclear contors, frequent multinucleation, vesicular chromatin, and prominent eosinophilic nucleoli (Reed–Sternberg cells) (Fig. 2). The atypical cells were positive for CD30, CD15, and PAX-5 and negative for CD2, CD4, CD20, CD45, and ALK-1 (Fig. 3). An EBER in situ hybridization stain was negative in the neoplastic cells. The morphology and immunohistochemistry were diagnostic for a nodular sclerosis classical Hodgkin lymphoma, syncytial variant, and in apparent contradiction with the immunophenotypic diagnosis of a T-cell lymphoproliferative disorder indicated by flow cytometry.

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Figure 1. Flow cytometric analysis showing the predominant cell population in red. Normal B lymphocytes are in blue, CD8(+) T-cells are in yellow, and remaining T-cells are in green.

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Figure 2. Atypical cells in the flow cytometry cytospin preparation (left) and in histologic sections (right).

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Figure 3. Immunohistochemical stains showing CD30, CD15, and PAX-5 expression in the atypical cells, which are also CD45(−).

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This apparent discrepancy has been addressed in the literature in two elegant immunophenotypic studies that have demonstrated the feasibility of diagnosing classical Hodgkin lymphoma by 9- or 10-color flow cytometry (1, 2). In general practice, routine flow cytometry is hampered in detecting neoplastic Hodgkin and Reed–Sternberg (HRS) cells, due to their infrequent occurrence in the analytic specimens (<1% of leukocytes), due to downregulation of B-cell surface antigens, and due to their unusual immunophenotype, which makes them difficult to isolate in a reactive cellular background. However, multicolor assays using a combination of antibodies targeted to HRS antigens (CD15, CD30, CD40, CD71, and CD95), in addition to CD19, CD20, CD3, CD5, CD64, and CD65, were able to identify HRS cells with high sensitivity and specificity in clinical specimens. Of note, a significant subset of cases demonstrated expression of T-cell antigens (CD3 and CD5) and bright CD45 on HRS cells, due to T-cells forming rosettes around the HRS cells. The resulting HRS-T cell aggregates had higher forward scatter and an apparent T-cell immunophenotype, which disappeared upon incubation with blocking antibodies (against CD2, LFA-1, CD54, or CD58) that disrupted this interaction.

The present case is an extreme example of this interaction between HRS cells and T-cells, visible morphologically in the flow cytometry cytospin by the presence of numerous T cell-HRS rosettes. The resulting hybrid immunophenotype is a combination of antigen expression contributed by HRS cells (CD30) and T-cells (CD2, CD3, CD4, CD5, CD7, CD38, and CD45). It is important to note that, when examining the T-cells with this observation in mind, the only unusual feature about them would be the elevated CD4:CD8 ratio, whereas other T-cell antigens are being expressed at normal levels. In contrast, immunophenotypic studies of T-cell lymphoproliferative disorders have shown consistent aberrant expression in several T-cell antigens in nearly all neoplastic cases, when compared to reactive conditions (3). For practical purposes, several studies have reported an elevated T-cell CD4:CD8 ratio in lymphocytes infiltrating classical Hodgkin lymphoma cases (4, 5), and a recent report has also noted an overexpression of CD7 in classical Hodgkin lymphoma-infiltrating T lymphocytes (6). More recently, a single 6-color antibody combination has been shown to have a sensitivity and specificity of 85.4% and 99.7%, respectively, for diagnosing classical Hodgkin lymphoma in lymph nodes (7).

In summary, flow cytometric detection of classical Hodgkin lymphoma cells is technically feasible using 6-color immunophenotyping, and may be applicable in common clinical practice in select cases. The phenomenon of HRS rosetting by T-cells can impart an apparent T-cell immunophenotype to the HRS cells, which, in extreme cases, may suggest an incorrect pathologic classification in the absence of morphologic and immunohistochemical data.

CASE 2 DIAGNOSIS: Classical Hodgkin Lymphoma

Acknowledgements

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED
  7. Supporting Information

The author acknowledges Alexandra M. Harrington, M.D. and Steven H. Kroft, M.D. for helpful discussions.

LITERATURE CITED

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED
  7. Supporting Information

Supporting Information

  1. Top of page
  2. CASE HISTORY
  3. FLOW CYTOMETRIC STUDIES
  4. DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_20598_sm_suppinfo1.fcs881KSupporting Information 1
CYTO_20598_sm_suppinfo2.fcs881KSupporting Information 1
CYTO_20598_sm_suppinfoCase2.pdf722KSupporting Information Case 2

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