Case report

A 75-year-old female with no significant medical history presented to the emergency department with a 6-week history of malaise, generalized pain, swelling over the upper chest, and left-sided back pain radiating to the left lower extremity. At presentation, a complete blood count (CBC) showed hemoglobin of 11.6 g/dL; mean corpuscular volume (MCV) of 92.5 fL; and platelet, white blood cell, and absolute eosinophil counts of 315 K/μL, 5.8 K/μL, and 0.48 cells/μL, respectively. The patient was hospitalized for further evaluation. CT-PET images were remarkable for a lytic lesion of the sternum, a left lower lobe lung nodule, multiple soft tissue masses in the subphrenic region adjacent to the kidneys and throughout the abdomen and pelvis, and lymphadenopathy involving multiple mesenteric nodes and a single retroaortic lymph node.

Fine needle aspiration and a core biopsy were obtained from the left-sided neck lymph node. Sections of the formalin-fixed, paraffin-embedded lymph node tissue demonstrated sheets of large lymphoid cell infiltrates with patchy necrosis and frequent apoptotic cells (Fig. 1a). Immunohistochemical stains were positive for BCL2 (> 70% of tumor cells) and MYC (> 90% of tumor cells) (data not shown). Greater than 90% of the tumor cells were positive for the immunohistochemical proliferation marker, Ki-67 (data not shown). Cytogenetic analysis using FISH indicated a MYC[8q24]/IGH[14q32] fusion and a 3’BCL2/5’BCL2 rearrangement. No rearrangements of the BCL-6, Ig Lambda, or Ig kappa genes were observed. Flow cytometry of the fine needle aspirate was performed using a FACSCanto II flow cytometer (BD Biosciences). Briefly, the sample was adjusted to a cell concentration of approximately 10.0 × 103 cells/μL. Processed cells were incubated with monoclonal antibodies using the following 8 color combinations: kappa-FITC/lambda-PE/CD34-PerCP-Cy5.5/CD10-PE-Cy7/CD5-APC/CD19-APC-H7/CD20-V450/CD45-V500c; CD103-FITC/CD11c-PE/CD22-PerCP-Cy5.5/CD38-PE-Cy7/CD23-APC/CD3-APC-H7/FMC-7-V450/CD45-V500c; and CD2-FITC/CD7-PE/CD4-PerCP-Cy5.5/CD56-PE-Cy7/CD5-APC/CD3-APC-H7/CD8-V450/CD45-V500c. Analysis was performed with FACSDiva software (BD Biosciences). Flow cytometric analysis revealed an immunoglobulin kappa light chain restricted B cell population expressing CD10, CD19, and CD20 (Fig. 2a). The lymphoma exhibited kappa light chain gene restriction. Cytogenetic studies by FISH analysis identified a MYC[8q24]/IGH[14q32] fusion and a 3’BCL2/5’BCL2 rearrangement (data not shown). The diagnosis of high-grade B cell lymphoma with MYC and BCL2 rearrangements (HGBCL) was rendered.

Fig. 1
figure 1

Histologic findings. a Histologic section of the H&E-stained cervical lymph node shows diffuse sheets of large lymphoid cells with prominent nucleoli and a high apoptotic rate. ×500 magnification. b Cytologic evaluation of cells obtained from pleural fluid demonstrates prominent nucleoli and a high apoptotic rate. The cells are morphologically similar to the cells evaluated in the left neck lymph node. Papanicolaou stain, ×500 magnification

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Fig. 2
figure 2

a Flow cytometric evaluation of the cervical lymph node shows a monotypic B cell population, positive with CD19, CD20, CD10, and surface kappa light chain. b Flow cytometric evaluation of the pleural fluid shows a monotypic B cell population with CD19, CD20, and CD10 positive immunophenotype, however, with lambda light chain restriction. Repeated flow cytometric analysis of the pleural fluid using different light chain antibodies confirmed the discordant light chain expression (data not shown)

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Two weeks after her initial presentation, the patient experienced shortness of breath secondary to a right pleural effusion. Sampling of the pleural fluid revealed atypical lymphoid cells that were morphologically similar to the lymphoma cells of the left neck lymph node (Fig. 1B). FISH analysis of the pleural fluid cells also identified a MYC[8q24]/IGH[14q32] fusion and a 3’BCL2/5’BCL2 rearrangement that was similar to the previously analyzed left neck lymph node cells. The pleural fluid cells were further analyzed by flow cytometry and revealed a monotypic B cell population with an immunophenotype that resembled the neck lymph node cells (CD10+, CD19+, CD20+), yet exhibited an immunoglobulin lambda light chain restriction (Fig. 2b). To rule out a laboratory error, flow cytometric analysis of lambda and kappa light chains of the pleural fluid was repeated using a different set of antibodies, which produced similar results, confirming lambda light chain restriction (data not shown).

In order to investigate the relatedness between the neck lymph node and pleural fluid B cell populations, B cell gene rearrangement analysis of the Ig heavy and Ig kappa light chain genes was performed by PCR using previously reported techniques [1]. Briefly, DNA was extracted from the left neck lymph node and pelleted pleural fluid, respectively, followed by PCR amplification using oligonucleotide primers specific for Ig Heavy and kappa light chain genes. Amplified products were separated by electrophoresis and evaluated for the presence of clonal gene rearrangements. Clonal B cell gene rearrangements of the immunoglobulin heavy chain were highly similar between the left neck lymph node and pleural fluid B cell populations (Fig. 3). Immunoglobulin kappa light chain clonal gene rearrangements were also similar between left neck lymph node and pleural fluid B cells for all gene segments evaluated (Fig. 3), suggesting a clonal relatedness between the two B cell populations. It should be noted, however, that although the Ig heavy and kappa light chains rearrangements were highly similar in size for the two B cell populations, the amplicons were not sequenced.

Fig. 3
figure 3

Comparative Immunoglobulin heavy chain gene and kappa light chain gene rearrangement study by PCR and capillary electrophoresis demonstrate highly similar clonal peaks in the pleural fluid and left neck lymph node

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The patient underwent R-EPOCH (rituximab, etoposide phosphate, prednisone, vincristine sulfate, cyclophosphamide, and doxorubicin hydrochloride) chemotherapy for 23 days before succumbing to her disease.

In addition to this case, a literature review revealed seven previously reported cases of B cell lymphoma with an immunoglobulin light chain switch. Table 1 summarizes the demographic, pathologic, and molecular features of the previously published cases alongside the current case. The B cell lymphomas with noted light chain switch occurred in the adult population (range 14–75 years; mean 54 years). Three of the cases detected the switch during a relapse [2,3,4]. The time to relapse ranged from 21 months to 18 years. With the exception of one reported case, the relapse was noted to have an immunoglobulin light chain switch from kappa to lambda. Three cases (including the current case) identified distinct populations with respective kappa and lambda light chain restriction at initial presentation [5, 6]. The subtypes of the B cell lymphomas included a follicular lymphoma which relapsed as a follicular lymphoma; another follicular lymphoma which relapsed as a HGBCL; a post-transplant lymphoproliferative disorder (PTLD), EBV+, which relapsed as relapsed as another PTLD, EBV+; a B cell lymphoma, kappa light chain restricted, with a concomitant lambda light chain restricted plasmacytoma with the same immunoglobulin gene rearrangement; a chronic lymphocytic leukemia (CLL), lambda light chain restricted with concomitant DLBCL, kappa light chain restricted; a CLL kappa light chain restricted relapsed as DLBCL with lambda light chain restriction; and a HGBCL with kappa light chain restriction and concomitant HGBCL with lambda light chain restriction (the current reported case). In 3 of the 5 cases, molecular methods confirmed the relatedness of the lymphomas with discordant light chain expression, including immunoglobulin heavy chain gene (IGH) PCR and comparative sequence analysis in 3 cases, and IGH and Ig kappa gene PCR in one case. A comparative analysis of the cytogenetic findings was also performed in our case.

Table 1 Reported cases of discordant light chain expression
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Discussion

We report a case of a high-grade B cell lymphoma with MYC and BCL2 rearrangements (HGBCL) with discordant light chain expression at different anatomic sites. The assumption of relatedness of the lymphoma in the lymph node and in the pleural fluid is based on a combination of morphologic, immunophenotypic, cytogenetic, and molecular findings. The morphologic features of the lymphoma cells are similar in the lymph node and pleural fluid, respectively. The flow cytometric properties are also very much in overlap, except for the discordant light chain expression. Most importantly, both lymphomas carry a MYC gene and a BCL2 gene rearrangement. HGBCL with MYC and BCL2 rearrangements (“double hit lymphoma”) is a very rare entity. The probability of one patient develo** two independent double hit lymphomas at two different anatomic sites concomitantly is very low and has not been ever reported. In the context of identical cytogenetic findings, the similarity of the amplicons in B cell IGH and kappa gene rearrangement PCR studies supports the relatedness. In our current case, we were not able to directly compare the rearranged immunoglobulin gene PCR amplicons by sequencing. While we acknowledge that this is a shortcoming of our study, since identical size amplicons do not necessarily indicate a clonal relationship, we feel that overall there is substantial evidence to support that the two B cell lymphomas represent the same disease.

Few cases of B cell lymphomas with discordant light chain expression have been previously reported in the literature. These cases challenge our understanding of the maturation and commitment of B lymphocytes, and they give rise to questions about the molecular steps during B cell development.

The discordant light chain expression observed in these cases should include the possibility of two distinct B cell neoplasms. Although two cases (Table 1: cases 5 and 6) consisted of B cell lymphomas with discrepant light chains, further analysis by PCR-based gene rearrangement studies revealed the malignancies to be completely unrelated [6]. Both of these cases also establish that unrelated B cell neoplasms may have differing morphology; however, one should recognize that varying morphology can also occur in clonally related B cell neoplasms (Table 1: case 4) emphasizing the need for genetic analysis if discordant light chain restriction is detected [5, 6].

Another explanation for the discordant light chain expression observed in these cases (a hypothesis that aligns with our current understanding of B cell development) is that a common precursor gives rise to separate kappa and lambda expressing subclones following the initial immunoglobulin heavy chain rearrangement. The B cell precursors would therefore give rise to two B cell populations with identical immunoglobulin heavy chains but are distinguished from each other due to their light chain restriction. This is certainly plausible in case 3, which is characterized by a post-transplantation lymphoproliferative disorder (PTLD) followed by recurrence of a clonally related PTLD [4]. Epstein-Barr Virus (EBV)-encoded RNA was expressed at the initial PTLD diagnosis, with undetectable EBV DNA levels during remission and increased during relapse, suggesting that a virally infected ancestral B cell gave rise to subclones that ultimately underwent divergent evolution [4]. Although the scenario of a single precursor giving rise to separate kappa and lambda subclones was also a proposed mechanism in case 4, this concept is challenged by two distinctly different histopathologic concomitant diagnoses, which would require the same precursor cell to undergo two different pathways to develop malignancy, and is instead more likely due to evolution of the B cell malignancy [5].

The observed discordant light chain features could also be observed due to the evolution of a mature B cell located within the germinal center of a peripheral lymph node. In this model, a lambda light chain restricted B cell lymphoma may evolve from neoplastic B cell that already expressed the kappa light chain. It has been traditionally thought that VDJ gene rearrangement is carried out by early B cells within the bone marrow under the influence of recombination activating gene (RAG)-1 and RAG-2 expression [7]. RAG-1 and RAG-2 gene expression is thought to be considered deactivated in mature B cells residing in peripheral tissues [8]. However, a combination of studies suggests RAG-1 and RAG-2 products to be active within germinal centers and contributing to VDJ recombination, capable of kappa and lambda chain rearrangements [9,10,11,12]. Interestingly, the immunoglobulin light chain switch observed in most of the cases was kappa to lambda, which demonstrate immunoglobulin kappa light chain restriction preceding lambda light chain restriction. Case 5 is one of the exceptions to the lambda to kappa light chain switch, however, that can be explained by the clonal unrelatedness of the two malignancies [6]. Additionally, the lambda to kappa switch noted on case 7 occurred in a pediatric case of recurrent follicular lymphoma. The clonal relatedness was not established and therefore presents a challenge of determining whether the kappa to lambda evolution is a notable feature of discordant light chain expression in clonally related B cell malignancies. The case, however, featured a single copy loss of chromosome 5 at band 5p14.3 that was also present in several family members and a family history of malignancy, including DLBCL, suggesting that the diagnoses of follicular lymphoma could have been due to two unrelated neoplasms [6]. In the case of recurrent follicular lymphoma (Table 1: case 1), both malignancies shared somatic mutations in a unique VH (CDR IIIH) segment as well as breakpoint sequences of a t(14:18) translocation establishing clonal relatedness [2]. Interestingly, Kobrin et al. went further in their analysis and utilized a PCR strategy to amplify a tumor-specific Vλ gene, which was present in cells evaluated at recurrence as well as the initial diagnosis [2]. Together, these findings suggest the evolutionary event to occur at the level of the germinal center.

The timing of presentation and localization for the two B cell lymphomas within our case may coincide with the immunoglobulin light chain switch. This could be explained by a malignant B cell clone identified in the left neck lymph node traveling to another site and undergoing conversion to lambda light chain restriction followed by subclonal expansion. For the patient in this case report, the lambda light chain restricted subclonal expansion could have taken place within either a thoracic cavity node or the enlarged retroaortic lymph node noted at the time of her initial presentation.

Finally, are external contributors, such as chemotherapy, responsible for the light chain switch? Several of the cases reported in the literature hint to genetic alterations in B cells following treatment with rituximab [3, 4, 13, 14]. Yet half of the cases, including our case, either have no previous exposure to rituximab or were diagnosed concomitantly, suggesting chemotherapy to be a less likely culprit for these particular B cell alterations and may instead result either from the array of mechanisms previously discussed. However, due to the limited number of cases, the role of chemotherapy in the development of B cell malignancy should continue to be investigated.

In summary, B cell lymphomas with discordant light chain expression are extremely uncommon, and they raise new questions regarding the current understanding of B cell development. The few reports in the literature, including this case, highlight the importance of integrating morphologic, immunophenotypic, cytogenetic, and molecular results into the diagnosis. Flow cytometry has a uniquely important role in identifying B cell lymphomas with discordant light chain expression in the same patient. Flow cytometrists should be aware of this rare phenomenon. At the same time, the flow cytometry laboratory should approach the observation of discordant light chain expression with caution and assure the accuracy of the finding by repeated testing using alternative kappa and lambda light chain reagents. From the clinical point of view, it may be critical to establish the relatedness of the two B cell lymphomas, as opposed to the rise of two independent B cell lymphomas in the same patient, since these two scenarios may require different treatment. It would be important to recognize B cell lymphomas with discordant light chain expression and secure them for additional cytogenetic and molecular studies, for they may reveal new information in B cell lymphoma pathogenesis and, ultimately, may guide treatment in the future.