Molecular genetic data favoring a sequential clonal transformation of a large B cell lymphoma into an anaplastic large T cell lymphoma, ALK-negative
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A large B cell lymphoma with monoclonal rearranged, sequentially identical TRG and near identical IGH genes twice relapsed as an anaplastic large T cell lymphoma, ALK-negative. While these IGH and TRG rearrangements were also detected in both relapsed lymphomas, near identical IGK rearrangement was present in the primary and the first relapse, though not in the second relapse, and identical TRB rearrangement was detected only in both relapses, not in the primary. Normal T lymphocytes were the only T cell component found in the primary lymphoma, thus excluding a possibility that the primary lymphoma was a classic composite B and T cell lymphoma. Our data, particularly, the presence of sequentially identical monoclonal TRG and near identical IGH rearrangement in the primary and both relapsed lymphomas, favor a gradual transformation of a B cell lymphoma into a T cell lymphoma. In our opinion, this previously not described phenomenon likely represents a composite lymphoma in statu nascendi.
KeywordLarge B cell lymphoma T lymphocytes IGK rearrangement
Rare cases of composite lymphoma composed of neoplastic B cell and T cells [1, 2, 3, 4, 5, 6, 7, 8] as well as simultaneous or sequential development of B cell and T cell lymphoma in the same patient [9, 10] point toward the possibility that rare lymphomas undergo lineage infidelity with resulting clonal development of a T cell lymphoma from a B cell lymphoma and vice versa. A recent large flow cytometry study revealed that lymphomas composed of neoplastic B and T cells constituted mere 0.4 % of all studied lymphomas . Due to technical limitations, this development has so far not been sufficiently analyzed. Molecular genetic analysis of the case that we present in this article delineates a sequential transformation of a B cell lymphoma into a T cell lymphoma. A similar case was not, to the best of our knowledge, previously described.
A 53-year-old white man presented for evaluation of a retroperitoneal mass that was detected in April 2008 in another hospital. A confluent paraortal and left inguinal adenopathy was confirmed by computed tomography. Six cycles of CHOP–Rituxan therapy (dexamethasone, doxorubicin, vincristine, cyclophosphamide, and rituximab) resulted in complete remission. He developed a relapse in the form of a right axillary lymphadenopathy and moderate to marked splenomegaly in February 2011. Five cycles of salvage ICE chemotherapy (ifosfamide, carboplatin, etoposide) led to remission. He developed a second relapse (generalized lymphadenopathy and progressive hepatosplenomegaly) in July 2012. His course became complicated by neutropenic fever, respiratory distress, and encephalopathy. He expired a few weeks later. Generalized lymphadenopathy, marked splenomegaly, extensive involvement of portal tracts, and multifocal lymphomatous involvement of the kidneys and bone marrow were found during autopsy.
Materials and methods
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections. Antibodies against the following epitopes were used: CD2, CD4, CD5, CD7, CD8, CD15, CD30, CD43, CD45, CD45RO, CD138, MUM1, EMA, ALK-1, cyclin D1 (Dako, Carpinteria, CA, USA), CD10, CD20, CD22, CD79a, bcl-2, bcl-6, Pax-5, BOB.1, OCT-2, Ki-67, TdT (Leica/Novocastra Microsystems, Buffalo Grove, IL, USA), CD163, EBV-LMP1 (Cell Marque, Rocklin, CA, USA), CXCL13 (R&D, Minneapolis, MN, USA), and C-MYC (Ventana Medical Systems, Tucson, AZ, USA). The antibodies were monoclonal except for the antibody against CXCL13 that was polyclonal. The slides to be immunostained for CD15, CD20, CD4, CD79a, CXCL13, and Ki-67 were treated by EnVision™ FLEX Target retrieval solution low pH (Dako), and the other slides were treated by EnVision™ FLEX Target retrieval solution high pH (Dako).
The following antibodies were utilized for flow cytometry analysis: anti-CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD16, CD19, CD20, CD23, CD30, CD33, CD56, FMC7, kappa, lambda, TCRα/β, and TCRγ/δ (Becton-Dickinson, San Jose, CA, USA).
The analysis was performed in two laboratories, Temple and Pilsen. The DNA was extracted from formalin-fixed, paraffin-embedded tissue using the Qiagen QIAamp Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocols in the Temple laboratory and NucleoSpin Tissue Kit (Macherey Nagel, Puren, Germany) in the Pilsen laboratory. In the Temple laboratory, the DNA was amplified by Invivoscribe Technologies (San Diego, CA, USA) kits targeting IGH, IGK, IGL, TRG, and TRB. The amplicons were detected on the ABI 3500 genetic analyzer (Life Technologies) using differential fluorescence technology. Integrity of DNA was verified in the Pilsen laboratory by PCR with BIOMED-2 control primer set (Generi Biotech, Hradec Kralove, Czech Republic) amplifying 100–600-bp range of fragments. The clonality analysis of TRG and IGH, and IGK and IGL was performed there with BIOMED-2 multiplex PCR system followed by fragment analysis of amplicons on ABI3130xI Genetic analyzer (Life Technologies) . Appropriate positive, negative, and contamination controls as well as the control ladder were run alongside the patient sample in both laboratories. Amplicons with one or two prominent peaks within the valid size range were considered to be monoclonal. The monoclonal peaks were re-amplified in the Pilsen laboratory with unlabeled “peak”-specific primer sets and purified and both strands sequenced using Bid Dye Terminator Sequencing kit (Life Technologies). The sequences were compared using in silico algorithms for comparing IG and TR nucleotide sequences [12, 13].
Deep sequencing for TRG and IGH was performed in the Temple laboratory using LymphoTrack® IGH and TRG assays (Invivoscribe Technologies, Carlsbad, CA) on the Ion Torrent PGM (Life Technologies). Multiplex master mix for IGH targets the conserved framework region 1 (FR1) within the VH and the JH regions, while multiplex master mix for TRG targets all conserved regions within the V and the J gene regions described in lymphoid malignancies. Primers included in the master mixes are designed with Life Technologies (Thermo Fisher) adapters and contain 12 different indices per kit. After routine DNA extraction and PCR amplification with primers and barcode adaptors onto the Ion Sphere Particles (ISPs), template-positive ISPs were subjected to next-generation sequencing (NGS). This was performed on Ion 316 chips using the Ion PGM™ Sequencing 200 kit v2 and the Ion Torrent PGM (Life Technologies). NGS data were analyzed using the LymphoTrack PGM Software package, and V–J sequence frequency plots as well as relative V–J usage plots were presented.
Molecular genetic study—polymerase chain reaction/clonal analysis
The IGH and TRG genes were monoclonally rearranged in the primary and both relapses. The monoclonal rearrangement of the IGH gene was detected only by deep sequencing. When analyzed in the Pilsen laboratory, two monoclonal TRG peaks, 218 and 243 bp, were found in the primary and both relapses. It appears that rearrangement of the same TRG region was detected in both laboratories. The 218-bp peak was found in the Vglf–Jg1.3_2.3 region (Vg2-8–J1/J2). The 243-bp peak, found in the Vglf–Jg1.1_2.1 region (Vg2-8–JP1/JP2), resulted from rearrangement of Vg4 and JP2 segments. The 218-bp peak was sequenced only from the second relapsed lymphoma and represents a nonproductive allele that most probably arose during bi-allelic TRG rearrangement . When analyzed in the Temple laboratory, a 73-bp TRG peak was found in the primary and both relapses and both relapses also contained an additional 252-bp monoclonal peak. The 73-bp peak was also detected in a 4-mm2 laser capture microdissected section of the primary that contained approximately 5600 cells (840 T lymphocytes). The 73-bp peak was low in the primary, became stronger in the first relapsed, and strong in the second relapsed lymphomas. The 73-bp peak represents the Vg1-8,9–Jg1/2, and the 252 peak represents the prior Alt.Vg–Jg1/2 regions of the TRG.
Sequencing of the TRG and IGK amplicons was performed in the Pilsen laboratory, and the sequences there were compared by IgBLAST and BLAST [12, 13]. The 243-bp TRG peak remained sequentially identical in the primary and both relapsed lymphomas and arose from the TRGV4*01/TRGJP2*01 junction. The 218-bp peak of the second relapse revealed joining of the TRGV2*02 and of the TRGJ1*02 or TRGJ1*01 or TRGJ2*01 segment.
Presence of identical clonal peak of TRG and near identical clonal peak of IGH in the primary large B cell lymphoma and both relapses, a decrease in intensity of the IGH peaks in both relapses that was coinciding with an increase in the TRG peak in the second relapse, presence of clonally rearranged TRB only in both relapses, and a loss of monoclonal IGK peak in the second relapse seem to indicate that the transformation of B cell lymphoma into T cell lymphoma was a gradual process. Sequentially identical TRG and near identical IGH monoclonal rearrangement in all three lymphomas provides the strongest argument in favor of their clonal relatedness. The number of T cells in the primary was estimated by flow cytometry to represent 15 % of the cells. Because presence of at least 5 % of cells with monoclonal TRG rearrangement is necessary for their detection by polymerase chain reaction in our laboratory, at least a third of the 840 T cells expected to be present in the microdissected portion would have to be positive for the rearrangement and to be, by implication, morphologically abnormal. Thus, the failure to detect a neoplastic T cell component in the primary B cell lymphoma by histology, immunohistochemistry, and flow cytometry does not support a possibility that this lymphoma contained two separate, fully developed, neoplastic components, a B cell lymphoma and a T cell lymphoma from the beginning and thus represented a classic composite B cell and T cell lymphoma. On the other hand, the data favor an early stage of development of composite lymphoma (composite lymphoma in statu nascendi). This conclusion is also supported by very low frequency of B lymphocytes and failure to detect any residual B cell lymphoma in the second relapse. It seems that the primary large B cell lymphoma contained a subpopulation of neoplastic B cells that were undergoing genetic/epigenetic changes representing early stage of transdifferentiation toward T cell lymphoma. These cells then continued the process of transdifferentiation, after eradication of original neoplastic B cells by chemotherapy, into T cell lymphoma.
Existence of clonal transformation of B cell lymphoma to T cell lymphoma is not necessarily surprising. It has been well established that under laboratory conditions, pro-B and pre-B cells can be reprogrammed into pluripotent stem cells and that even mature B cells can be converted into T cells by their dedifferentiation to uncommitted progenitors [16, 17, 18]. The chain of events that resulted in transformation of our case of large B cell lymphoma into anaplastic large T cell lymphoma is not known. It may be noteworthy that the Pax-5 gene, the essential regulator of B cell identity , was strongly expressed in the primary lymphoma but was not expressed in the relapsed lymphomas. We do not know whether this gene was lost or was epigenetically silenced in the recurrent lymphomas.
Although rare cases of clonal transdifferentiation of hematopoietic neoplasms were reported, their existence does not appear to be sufficiently recognized. They include transformation of lymphoid neoplasms into Langerhans cell neoplasms and histocytic sarcomas and chronic myeloid leukemia into T cell lymphoblastic lymphoma [20, 21, 22, 23, 24, 25].
We were initially unable to classify the first relapsed lymphoma. Although we favored a peripheral T cell lymphoma, the presence of CD15-positive, CD30-positive blasts as well as background of numerous T lymphocytes, many eosinophils and occasional plasma cells were raising a possibility of Hodgkin’s lymphoma. However, absence of Sternberg–Reed cells was a strong argument against this interpretation. Several cases of CD30-positive T cell lymphomas coexpressing CD15 were previously reported .
Phenomenon of lineage infidelity when monoclonally rearranged IG and TR genes coexist in the same cell has been well recognized. Its frequency varies from 5 to 10 % of well-differentiated B cell neoplasms, e.g., B cell chronic lymphocytic leukemia to 70 % of less-differentiated B cell neoplasms, e.g., B acute lymphoblastic leukemia/lymphoma. Several studies of relapsing lymphomas and B acute lymphoblastic leukemias [27, 28, 29, 30, 31, 32, 33] revealed that the monoclonal rearrangements of the IGH and, to a lesser extent, TR genes can be capricious. These genes can disappear at relapse, and the primary neoplasms can be overgrown by subclones with divergent rearrangement patterns. Similar changes likely took place during the course of transformation of our case.
Although gradual transformation of B cell lymphoma into T cell lymphoma is a rarity, this phenomenon is likely to be of clinical significance in individual cases.
Conflict of interest
The authors declare that they have no conflict of interest.
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