Transformation of Small B-Cell Lymphoma Into Large Cell [CD30.sup.+], [CD4.sup.+], Epstein-Barr Virus-Negative Lymphoma.
In this article, we present 2 separate but similar cases of [CD30.sup.+] B-cell lymphomas that do not fit into any of the categories described above. Both patients presented with a morphologically high-grade lymphoma whose immuno phenotype ([CD30.sup.+], [CD4.sup.+], EBV-, B-cell lineage markers with negative or focal and weak staining) led to an initial suspicion of ALCL in both cases. However, both patients had a history of prior low-grade follicular lymphoma, and molecular studies demonstrated the same clonal pattern of immunoglobulin heavy chain gene (IGH@) rearrangements in both the transformed lymphomas and their corresponding original low-grade B-cell lymphomas. To our knowledge, these cases are the first to be reported that demonstrate this particular combination of morphology (anaplastic large cells) and immunophenotype (CD30 strongly positive; negative for other markers including Epstein-Barr virus-encoded small RNA [EBER], histiocytic/ dendritic cell markers, anaplastic lymphoma kinase [ALK], and CD138; and B-cell markers negative or weak and focal) in a malignancy that can be incontrovertibly shown on molecular grounds to be a transformed follicular lymphoma. These cases are relevant not only as a further example of the plasticity of tumors of B-cell origin, but also as a way to highlight a potential diagnostic pitfall, since both were nearly given the formal diagnosis of ALCL before knowledge of the patients' antecedent low-grade lymphomas prompted further immunohistochemical and molecular studies.
Patient 1 is a 50-year-old man who presented in August 2008 with inguinal lymphadenopathy, proven by biopsy of an inguinal lymph node to be low-grade follicular lymphoma. He was subsequently treated with multiagent chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP]), followed by 2 cycles of rituximab and bendamustine. A computed tomography scan performed subsequent to this therapy demonstrated continuing generalized lymphadenopathy, without evidence of interval decrease, compared to initial images. Repeated biopsy was performed 18 months later, demonstrating the presence of a large cell lymphoma. All original biopsies were performed at an outside institution, and all material, including paraffin-embedded tissue blocks, were received for review at Vanderbilt University Medical Center (VUMC) in Nashville, Tennessee, as part of the patient's plan of care for treatment of large cell lymphoma.
Patient 2 is a 50-year-old man who presented with left inguinal and pelvic lymphadenopathy, proven by biopsy to be low-grade follicular lymphoma with bony lesions. The patient underwent palliative radiotherapy, with subsequent progression of disease to nodes (right pectoral and supraclavicular) outside of the radiation field. Excisional biopsy of a right cervical node was performed 2 years after initial diagnosis, documenting the presence of a large cell lymphoma. All biopsies were performed at Cooper University Hospital (CUH) in Camden, New Jersey.
Follow-up clinical data were unavailable for both patients after pathology review.
MATERIALS AND METHODS
Morphologic Studies, Immunohistochemistry, and In Situ Hybridization
The 2 patients were seen independently at separate institutions: patient 1 at VUMC and patient 2 at CUH, with substantially overlapping but independent workups performed. Hematoxylin-eosin (H&E)-stained slides were cut from paraffin-embedded tissue blocks (from an outside institution for patient 1, with studies performed on 5-im sections; in-house blocks for patient 2, with studies performed on 4-im sections). Immunohistochemical stains and in situ hybridization (ISH) studies were performed at each institution separately, as summarized in Table 1.
For both patients, DNA isolation was performed from paraffin-embedded tissue blocks by using commercially available kits as summarized in Table 1. For patient 1, IGH@ VDJ rearrangements were tested by polymerase chain reaction (PCR), using primers specific for consensus sequences in the VHFRIII region of IGH@ and appropriate controls, (6) followed by separation on a 10% polyacrylamide gel and visualization with ethidium bromide. For analysis of TRG@ VJ rearrangements, PCR amplification of multiple sites in the T-cell receptor region was performed with previously reported primers and modified reaction conditions. (7) Amplicons were radioactively labeled, separated by using a 6% denaturing polyacrylamide gel, and visualized by using autoradiography. BCL2 gene rearrangement studies were performed with primers specific for the [J.sub.H] region of IGH@ and for the major breakpoint region (MBR) and minor cluster region (mcr) of the BCL2 locus. (8) Polymerase chain reaction products were separated on a 0.7% agarose gel, analyzed by Southern blot, and hybridized to BCL2specific [sup.32]P-labeled specific probes. (8) For patient 2, IGH@ and TRG@ rearrangement studies were performed with commercially available kits, as summarized in Table 1. BCL2 gene rearrangement studies were performed by using quantitative real-time PCR assays on an ABI7300 thermocycler (Applied Biosystems, Foster City, California) with specific primers for the BCL2 MBR-[J.sub.H] and the BCL2 mcr-[J.sub.H] rearrangement regions, according to the BIOMED-2 Concerted Action protocol. (9)
Review at VUMC of the H&E-stained slides for the original low-grade lymphoma of patient 1 revealed a lymph node entirely effaced by a population of small lymphocytes in a predominantly diffuse pattern, with focal organization into round follicles with absent zonation, markedly attenuated mantle zones, and lack of tingible body macrophages. The lymphocytic infiltrate was composed predominantly of a monotonous population of small centrocytes with occasional intermediate-sized cells with small nucleoli (Figure, A). Slides of recurrent lymphoma for patient 1 demonstrated complete effacement by sheets of large, abnormal cells with large oval to irregular nuclei, vesicular chromatin, single central nucleoli, and abundant eosinophilic cytoplasm (Figure, B). Focal areas of sclerosis and coagulative necrosis were noted.
Review at CUH of H&E-stained slides from the low-grade lymphoma of patient 2 revealed a lymph node replaced by a population of small- to medium-sized centrocytes in a vaguely nodular pattern (Figure, C) without a significant population of large, irregular centroblasts or anaplastic cells. Slides of transformed lymphoma from the patient demonstrated a node partially replaced by a diffuse infiltrate of large cells with irregular nuclei, large oval to slightly irregular nuclei, vesicular chromatin, occasional prominent nucleoli, and abundant amounts of eosinophilic cytoplasm (Figure, D). Focal confluent necrosis was present.
In the transformed samples from both patients, there were no classic Reed-Sternberg or hallmark cells, and no morphologic evidence of persistent low-grade lymphoma.
Immunohistochemical Studies and In Situ Hybridization
The results of immunohistochemical staining, as well as in situ hybridization and molecular studies, on all 4 samples are summarized in Table 2. To summarize the most relevant findings, the initial lymphoma of patient 1 had a typical follicular lymphoma immunophenotype, expressing CD10, CD20, BCL2, and BCL6, and was negative for CD30 and EBER. The transformed lymphoma of patient 1 was diffusely and strongly positive for CD4 and CD30, negative for CD10 and BCL2, weakly and focally positive for CD79a, BCL6, and PAX5, and negative for CD20 (with a history of rituximab treatment). Additional markers, including EBER, CD138, ALK, and many T-cell and histiocytic/dendritic cell markers, all showed negativity (listed fully in Table 2).
The initial lymphoma of patient 2 expressed CD10, CD20, BCL6, and PAX5, and was negative for CD30, BCL2, and EBER. The transformed lymphoma of patient 2 was diffusely and strongly positive for CD4 and CD30, was focally positive for CD79a and PAX5, and was negative for CD10, CD20, CD22, BCL2, and BCL6. Additional markers, including EBER, ALK, and many T-cell and histiocytic/dendritic cell markers all showed negativity (listed fully in Table 2).
Molecular Genetic Studies
IGH@ rearrangement studies on samples from both patients revealed identical monoclonal bands for the original and transformed lymphomas in each case. TRG@ [gamma] rearrangement studies performed on both samples from each patient revealed no evidence of a monoclonal rearrangement. BCL2 rearrangement studies on patient 1 demonstrated an identical rearrangement involving MBR in both the original and transformed lymphomas. BCL2 rearrangement studies performed on both samples from patient 2 revealed no evidence of a t(14; 18) translocation in either the original or the transformed sample.
The original lymphoma of patient 1 was diagnosed as a follicular lymphoma, World Health Organization (WHO) grade 1 to 2 of 3, with a predominantly diffuse pattern. The transformed lymphoma of this patient was diagnosed at VUMC as large cell lymphoma, arising from transformation of the original follicular lymphoma; this corrected the initial diagnosis of ALCL that had been rendered at an outside institution without the benefit of a full set of immunohistochemical or genetic studies.
The original lymphoma of patient 2 was diagnosed as follicular lymphoma, WHO grade 1 to 2 of 3, with a predominantly follicular pattern. The clinical report noted that BCL2 negativity by immunohistochemistry can be seen in up to 15% of low-grade follicular lymphomas. (10) The transformed lymphoma in patient 2 was initially diagnosed as ALCL, but on further consultation and review, and with benefit of a full immunohistochemical and molecular workup, the diagnosis was amended to [CD30.sup.+] large cell lymphoma of B-cell origin.
We present here 2 cases of small B-cell lymphoma that each transformed into a lymphoma with aggressive features, including anaplastic morphology, with loss of many B-cell lineage-specific antigens as well as loss of germinal center markers. At the same time, both cases acquired diffuse and strong expression of CD30 and CD4. The loss of CD20 expression in patient 1 may be ascribed to rituximab administration; however, the significant loss of many other markers, including CD79a and PAX5, as well as other markers present in the original lymphoma, including CD10, BCL2, and BCL6, cannot be explained by rituximab effect. Patient 2 was never treated with rituximab and lost expression of CD20 along with many other B-cell markers. In both cases, T-cell and histiocytic/dendritic cell markers showed negativity, as did ISH for EBER (see Table 2 for a summary of findings). In the absence of any concomitant morphologic or immunophenotypic evidence of persistent low-grade lymphoma to suggest transformation, only the presence of identical IGH@ (and BCL2 in the case of patient 1) rearrangements permitted the unequivocal identification of large cell lymphomas as transformed B-cell lymphomas, clonally related to the previous low-grade disease.
Although strong and uniform CD30 expression may be seen in a variety of B-cell lymphomas, no other case reports in the literature document a combination of morphology and immunophenotype similar to that described here. The most well-known CD30+ B-cell neoplasm is classical Hodgkin lymphoma, with its characteristic morphologic appearance and immunophenotype, including expression of PAX5 and a few other B-cell markers, with only rare expression of T-cell markers. (11) Among the non-Hodgkin B-cell lymphomas, those associated with EBV or other viral infection may frequently express CD30 variably, consistent with EBV's known ability to upregulate CD30 expression in B-cells during infection, often showing bizarre, large cell morphology. However, our case fits neither the morphologic nor the immunophenotypic profile of classical Hodgkin lymphoma nor is it EBV positive.
While CD30 expression is well described as an uncommon event associated with transformation of follicular lymphoma, in all reported cases, CD30 expression is seen alongside normal expression of standard markers of B-cell or follicular center differentiation, including CD20, CD79a, PAX5, and BCL6. (4,5) Transformed follicular lymphomas can lose some B-cell-specific markers, ranging from the loss of CD20 positivity classically seen after treatment with rituximab to, more uncommonly, a wide-ranging loss of multiple B-cell markers. Loss of CD20 and other B-cell marker expression has been associated with more aggressive clinical behavior and poor prognosis. (12,13) However, this loss of B-cell markers has not previously been described in association with the uniform strong CD30 expression that was seen in our case.
Transformation of B-cell lymphomas into histiocytic/ dendritic cell sarcomas has been described in multiple case reports and case series. (2,3) These transformed neoplasms often have bizarre, anaplastic cytomorphology, and a significant subset of cases express CD30. Staining with B-cell markers is negative or weak, and there is no association with EBV. However, malignant cells in these cases all are positive for significant expression of histiocytic/dendritic cell markers, including CD1a, CD23, CD68, CD163, and S100. Our cases were negative for a wide range of markers that characterize histiocytes or dendritic cells.
In one case report, Ozdemirli et al (14) described the transformation of chronic lymphocytic leukemia to a large cell lymphoma with CD30 and CD4 expression and anaplastic morphology. This large cell lymphoma shares many clinical similarities with ours, including expression of CD30 and CD4 with loss of B-cell markers, but was strongly EBER positive, and occurred in the context of an EBV infection.
Both cases reported here were diffusely positive for CD4 in addition to their CD30 expression. Aberrant CD4 expression has been documented in a variety of B-cell lymphomas, including ALK-positive large B-cell lymphoma, diffuse large B-cell lymphoma, and classical Hodgkin lymphoma, in all cases in the context of an otherwise normal immunophenotype for that category of disease. (15) In the 2 cases reported here the CD4 positivity is remarkable less as an unusual occurrence in its own right, but more for the danger that coexpression of CD4 alongside CD30 might lead to misdiagnosis as an ALCL.
The 2 cases presented in this report demonstrate the novel finding of transformation of low-grade B-cell lymphoma to a [CD30.sup.+], [CD4.sup.+] large cell lymphoma with profound loss of B-cell lineage-specific markers, negativity of T-cell and histiocytic/dendritic cell lineage markers, and without any evidence of EBV infection. Although many cases have been described that match some elements of this presentation, we are unable to find any reports in the literature of cases with this particular combination of morphology and immuno-phenotype. More than just an unusual combination of markers, the particular phenotype seen in these cases represents an instructive danger to the practicing pathologist; both cases were temporarily misclassified as ALCL before each was reevaluated with full immunohistochemical and molecular genetic studies. In these cases, ancillary molecular genetic testing provides a vital key for rendering a definitive diagnosis.
Please Note: Illustration(s) are not available due to copyright restrictions.
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Aaron C. Shaver, MD, PhD; Ly Ma, MD; Cindy Vnencak-Jones, PhD; Roland S. Schwarting, MD; Kristina J. Fasig, MD; Annette S. Kim, MD, PhD
Accepted for publication August 8, 2013.
From the Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, Tennessee (Drs Shaver, Vnencak-Jones, Fasig, and Kim); and the Department of Pathology, Cooper University Hospital, Camden, New Jersey (Drs Ma and Schwarting).
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Annette S. Kim, MD, PhD, Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, 1301 Medical Center Dr, 4603A TVC, Nashville, TN 37232-5310 (e-mail: email@example.com).
Caption: Morphologic appearance of original and transformed lymphomas. Hematoxylin-eosin-stained slides from patient 1 (A and B) and patient 2 (C and D). Both original lymphomas (A and C) consisted of small, irregular lymphocytes with scant cytoplasm. The transformed lymphomas (B and D) were composed of large, anaplastic cells, with open chromatin, single central nucleoli, and abundant amounts of eosinophilic cytoplasm. No significant residual population of small abnormal lymphocytes was present in this specimen for either patient (original magnifications X400 [A through D]).
Table 1. Suppliers and Platforms for Materials and Methods Immunohistochemistry and Antibody Supplier In Situ Hybridization Patient 1: CD1a, CD3, CD20, CD79a, Ventana (Tucson, Arizona) CD138, ALK, EMA CD4, CD5, CD8, CD10, Leica-Microsystems CD23, CD30, CD68, (Bannockburn, Illinois) BCL2, BCL6, PAX5, S100 CD7, lysozyme Dako (Carpinteria, California) TIA1 Immunotech (Prague, Czech Republic) EBER Leica-Microsystems Patient 2: CD3, CD20, CD30, BCL2 Ventana CD4, BCL6 Cell Marque (Rocklin, California) PAX5 BioCare (Concord, California) CD10 Leica-Microsystems CD163 Novocastra (Bannockburn, Illinois) EBER Ventana Molecular Studies Kit/Apparatus Patient 1: DNA isolation QIAquick PCR Purification Kit Patient 2: DNA isolation QIAamp DNA Mini Kit IGH gene amplification Biomed-2 IGH primer set (Cat No. 1-101-0021) TCR gene amplification TCR gamma primer set (Cat No. 1-207-0011) Quantitative real-time PCR ABI7300 thermocycler Capillary electrophoresis ABI310 and ABI Prism GeneScan Immunohistochemistry and Stainer In Situ Hybridization Patient 1: CD1a, CD3, CD20, CD79a, Ventana BenchMark Ultra CD138, ALK, EMA CD4, CD5, CD8, CD10, Leica BondmaX CD23, CD30, CD68, BCL2, BCL6, PAX5, S100 CD7, lysozyme Dako Autostainer Plus TIA1 Dako Autostainer Plus EBER Leica BondmaX Patient 2: CD3, CD20, CD30, BCL2 BenchMark XT CD4, BCL6 BenchMark XT PAX5 BenchMark XT CD10 BenchMark XT CD163 BenchMark XT EBER BenchMark XT Molecular Studies Supplier Patient 1: DNA isolation Qiagen (Valencia, California) Patient 2: DNA isolation Qiagen IGH gene amplification InVivoScribe Technologies (San Diego, California) TCR gene amplification InVivoScribe Technologies Quantitative real-time PCR Applied Biosystems (Foster City, California) Capillary electrophoresis Applied Biosystems Abbreviations: ALK, anaplastic lymphoma kinase; EBER, Epstein-Barr virus-encoded small RNA; EMA, epithelial membrane antigen; IGH, immunoglobulin heavy chain; PCR, polymerase chain reaction; TCR, T cell receptor. Table 2. Morphologic, Immunophenotypic, and Genetic Features Patient 1 Initial Transformed Morphologic pattern Predominantly diffuse Diffuse Cytomorphology Small, low grade Anaplastic IHC, B-cell markers CD20 + (-) (a) CD22 ND ND CD79a ND Weak, focal PAX5 + Weak, focal IHC, T-cell markers CD3 - - CD4 - + CD5 (-) (b) - CD7 ND - CD8 ND - TIA1 ND - IHC, histiocytic/dendritic cell markers CD1a ND - CD23 - - CD68 ND - CD163 ND ND Lysozyme ND - S100 ND - IHC, additional markers CD10 + - CD30 - + CD43 (-) (b) (-) (b) CD138 ND - ALK ND - BCL2 + - BCL6 + Weak, focal EMA ND - MPO ND ND ISH for EBER - - Genetic studies ICH rearrangement Monoclonal band Monoclonal band, identical to original TCR rearrangement No monoclonal No monoclonal BCL2 rearrangement Positive, involving MBR Positive, involving MBR Patient 2 Initial Transformed Morphologic pattern Follicular Diffuse Cytomorphology Small, low grade Anaplastic IHC, B-cell markers CD20 + - CD22 ND - CD79a ND Weak, focal PAX5 + Moderate, subset IHC, T-cell markers CD3 - - CD4 - + CD5 - - CD7 ND ND CD8 - - TIA1 ND ND IHC, histiocytic/dendritic cell markers CD1a ND ND CD23 - - CD68 ND ND CD163 ND - Lysozyme ND ND S100 ND ND IHC, additional markers CD10 + - CD30 - + CD43 ND Subset CD138 ND ND ALK ND - BCL2 - - BCL6 + - EMA ND ND MPO ND - ISH for EBER - - Genetic studies ICH rearrangement Monoclonal band Monoclonal band, identical to original TCR rearrangement No monoclonal No monoclonal BCL2 rearrangement Negative Negative Abbreviations: ALK, anaplastic lymphoma kinase; EBER, Epstein-Barr virus-encoded small RNA; EMA, epithelial membrane antigen; IGH, immunoglobulin heavy chain; IHC, immunohistochemistry; ISH, in situ hybridization; MBR, major breakpoint region; MPO, myeloperoxidase; ND, test not performed on that specimen; TCR, T cell receptor; +, positive expression for that marker; -, absent expression for that marker. (a) Indicates prior treatment with rituximab. (b) Indicates a test performed by report at original referring institution, and not replicated.