Clonal analysis of a case with fluid overload-associated large B-cell lymphoma evolving from primary large B-cell lymphoma of the central nervous system

Abstract

A 62-year-old female was referred to our hospital because of left pleural effusion. She had a history of primary large B-cell lymphoma (LBCL) of the CNS (PCNSL), which was treated with chemoradiotherapy 14 years ago. Imaging studies revealed no other lesions. Pathologically, large atypical lymphoid cells were present in the pleural fluid and positive for CD20 and CD79a, while EBV and HHV-8 were negative. She was diagnosed with HHV8-negative effusion-based lymphoma (EBL) and achieved complete response after two cycles of rituximab monotherapy. We performed targeted-capture sequencing of lymphoma-associated genes using tumor samples from EBL and PCNSL, and detected the identical mutations in eight genes including MYD88 and CD79B. Identical arm-level copy number changes and focal deletions involving HLA-B, HLA-C, and CDKN2A, were also observed, suggesting clonal association between EBL and PCNSL. On the other hand, there were several lymphoma subtype-specific alterations, such as CARD11 and KMT2D mutations in PCNSL as well as PIM1 and PRDM1 mutations in EBL. LBCLs involving specific extranodal sites, such as CNS, testis, and adrenal gland, tend to recur in CNS, and harbors characteristic driver alterations, such as mutations in MYD88 and CD79B, similar to the MCD subtype of diffuse large B-cell lymphoma. Our genetic analysis suggests that in this case (i) PCNSL recurred as EBL or (ii) EBL developed from a common founder clone with PCNSL after long-term remission, providing insights into genetic basis underlying extranodal LBCL with CNS tropism.

Main

Fluid overload-associated large B-cell lymphoma (FO-LBCL), also known as Kaposi sarcoma-associated herpesvirus/human herpesvirus 8 (HHV8)-negative effusion-based lymphoma, is a new entity introduced in the fifth edition of the World Health Organization Classification of Haematolymphoid Tumours [1]. Like primary effusion lymphoma, FO-LBCL typically manifests as lymphomatous effusions within the body cavities, such as the pleural, pericardial, and abdominal cavities, with no detectable tumor masses. Although FO-LBCL mostly relapses within the body cavities, some cases recur in the extranodal sites, particularly the central nervous system (CNS) [2]. Here, we present a patient who developed FO-LBCL 14 years after achieving complete response (CR) of primary LBCL of the CNS (PCNSL). We performed targeted-capture sequencing (targeted-seq) analysis of both tumors and revealed their genetic relationships.

A 62-year-old female was referred to our hospital because of moderate left pleural effusion. She had a history of cerebellar PCNSL 14 years earlier and had been in sustained CR following initial treatment of high-dose methotrexate and whole-brain irradiation (45 Gy). Imaging examinations revealed a left pleural effusion without any other lesions (Fig. 1a and b). Pathological assessment of the pleural fluid cell block showed large atypical lymphoid cells that were positive for CD20 and CD79a, but negative for Epstein-Barr virus or HHV-8 (Fig. 1c–f). Consequently, she was diagnosed with FO-LBCL and achieved remission after two cycles of rituximab monotherapy combined with chest drainage.

Fig. 1.

Contrast-enhanced computed tomography (CT)/positron emission tomography (PET)-magnetic resonance imaging (MRI) of the chest and histopathology of pleural fluid cell block.(a and b) Contrast-enhanced thoracic CT (a) and PET-MRI (b) showing moderate left pleural effusion without any detectable tumor mass.(c–f) Histopathology of pleural fluid cells exhibiting CD20-positive pleomorphic large lymphoid cells (c and d) with negative staining for HHV-8 (e) and Epstein-Barr virus-encoded RNA in situ hybridization (EBER-ISH) (f), which are typical findings of FO-LBCL (×600 magnification).

To elucidate the genetic relationship between PCNSL and FO-LBCL in this case, we performed targeted-seq for a formalin-fixed paraffin-embedded (FFPE) tumor sample of PCNSL and a pleural fluid cell block sample of FO-LBCL as previously described [3].

Overall, we identified 12 single nucleotide variants (SNVs) in the PCNSL sample, while 23 SNVs and 2 insertions and deletions were detected in the FO-LBCL sample. Copy number (CN) analysis of the PCNSL sample revealed 4 arm-level CNAs including 2 gains and 2 losses and 5 focal CNAs including 2 amplifications and 3 deletions, while 8 arm-level CNAs including 5 gains and 3 losses and 6 focal deletions were present in the FO-LBCL sample. Among them, 8 mutations were detected in both PCNSL and FO-LBCL samples, such as gain-of-function mutations in MYD88 (p.L265P) and CD79B (p.Y196H) and loss-of-function mutations in TBL1XR1, HIST1H1E, and HLA-B (Fig. 2a). In addition, 4 arm-level CNAs (-6q, +9, +12, and -X) and 2 focal deletions of 6p21.33 (HLA-B and HLA-C) and 9p21.3 (CDKN2A) were observed in both samples. In addition, we identified an identical IGK clonotype in the FO-LBCL and PCNSL samples using MiXCR (version 4.3.2), suggesting these tumors originated from the same clone.

Fig. 2.

Clonal relationship between PCNSL and FO-LBCL. (a) Distribution of variant allele frequencies (VAFs) and types of mutations in PCNSL and FO-LBCL samples. b Two scenarios for clonal relationship between PCNSL and FO-LBCL.

On the other hand, four mutations, including gain-of-function mutations in CARD11 (p.R113Q) and loss-of-function mutations in KMT2D, and three focal CNAs were exclusively observed in PCNSL (Fig. 2a). By contrast, the FO-LBCL sample specifically harbored 17 mutations, including hypermutations in PIM1 and loss-of-function mutations in PRDM1, CD58, and BIRC3, 4 arm-level CNAs (-7p, +7q, +16, and +22), and 4 focal CNAs including a 6q21 homozygous deletion (encompassing PRDM1). In addition, a different HLA-B (p.T249A) mutation from shared one (stopgain SNV) was present only in FO-LBCL.

Here we report the first case who developed FO-LBCL after a history of PCNSL, showing a clonal relationship between them. Although most recurrences of PCNSL are confined to the CNS, some relapses occur in extranodal sites [4–6]. On the other hand, secondary CNS involvement is frequently observed in LBCL arising in extranodal sites [7]. These observations suggest that LBCL involving the extranodal sites tend to relapse in these sites, as shown in this case, and form a distinct group within LBCL.

Recent genetic studies have demonstrated that a molecular subtype named as MCD after MYD88 p.L265P and CD79B mutations, accounts for LBCL arising in the extranodal sites, such as PCNSL and primary testicular lymphoma [8]. The MCD subtype frequently involves extranodal sites, has a worse prognosis, exhibits an activated B-cell (ABC)–like phenotype, and harbors characteristic alterations, such as those in PIM1, HLA-B, TBL1XR1, and PRDM1, as detected in this case [8]. MYD88 p.L265P and CD79B p.Y196H mutations are gain-of-function mutations that are capable of constitutively activating the NF-κB pathway indispensable for the survival and proliferation of ABC-DLBCL cells [9]. In genetically engineered mouse models resembling the MCD subtype, including those harboring MYD88 p.L252P (murine equivalent of MYD88 p.L265P), the tumors involve extranodal regions [10, 11]. These observations underpin the possibility that genetic mechanisms characterizing the MCD subtype underlie the tropism for extranodal sites.

Although FO-LBCL typically shows the MCD subtype [12], its behavior appears to be more indolent, with some cases showing even spontaneous regression or cured only with rituximab monotherapy, as observed in this case. In PCNSL, treatment is intrinsically challenging due to the presence of the blood–brain barrier, which limits the penetration of therapeutic agents into the CNS [13]. Moreover, the immune-privileged nature of the CNS microenvironment promotes immune escape of tumor cells, thereby diminishing the effectiveness of treatment [14]. Thus, although subtype-specific alterations may influence the disease phenotype, its anatomical localization and microenvironment may have more impact on the difference of clinical manifestations between PCNSL and FO-LBCL in this case.

Our genetic analysis suggests two possible scenarios of tumor evolution in this case. One possibility is that FO-LBCL developed as a relapse of PCNSL, in which IKZF1, NFKB2, KMT2D, and CARD11 mutations are considered as subclonal (Fig. 2b). The other possibility is that both PCNSL and FO-LBCL developed from a common founder clone outside the CNS with the MCD subtype. In this scenario, CARD11 and KMT2D mutations may have allowed the founder clone to develop into PCNSL, while PRDM1, CD58, and BIRC3 mutations and so on are thought to enable the clone to manifest as FO-LBCL. A larger number of FO-LBCL-specific mutations are likely to reflect the longer latency until the onset of the disease.

In summary, we report a case who developed FO-LBCL after long-term remission of PCNSL, in which both lymphomas were clonally related. These observations provide insights into the genetic basis underlying extranodal LBCL with CNS tropism and have implications for their management strategies, particularly in monitoring and preventing the disease recurrence.

Authors’ contributions

K.I. and K.K. conceived and designed the study. R.H., S.F., W.M., Y.N., and K.I. managed the patient. A.M.M. performed the histopathological assessment. K.C., A.O., and Y.S. developed sequence data processing pipelines. Y.I. and Y.K. performed targeted-capture sequencing. Y.I., Y.K., and K.K. analyzed and interpreted the data. Y.I., Y.K., K.I., and K.K. wrote the initial draft. All authors reviewed and approved the final draft of the manuscript.

Funding

This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (JP21H05051), Japan Science and Technology Agency Moonshot R&D Program (JPMJMS2022-4), and Japan Agency for Medical Research and Development (JP24ck0106794h0002).

Data availability

The data presented in this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was performed in accordance with the Declaration of Helsinki and was approved by the National Cancer Center Institutional Review Board. We obtained written informed consent for publication in print and electronically from the patient.

Competing interests

Y.K. has received honorarium from Kyowa Kirin, Nippon Shinyaku, Daiichi Sankyo, and Takeda Pharmaceutical. S.F. has received honorarium from Janssen Pharmaceutical, Ono Pharmaceutical, Takeda Pharmaceutical, Amgen, Chugai, AbbVie, AstraZeneca, and Otsuka Pharmaceutical; has received research funding from Chugai Pharmaceutical, Loxo Oncology (Eli Lilly and Company), and Mitsubishi Tanabe Pharma. K.I. has received honorarium from Eli Lilly and Company, Merck Sharp & Dohme, Eisai, Chugai, Novartis, AbbVie, Ono Pharmaceutical, AstraZeneca, Kyowa Kirin, Bristol Myers Squibb, Takeda Pharmaceutical, Pfizer, Janssen Pharmaceutical, Daiichi Sankyo, and Symbio Pharmaceuticals; has received research funding from Merck Sharp & Dohme, Incyte, Eisai, AbbVie, Ono Pharmaceutical, AstraZeneca, Yakult, Kyowa Kirin, Loxo Oncology (Eli Lilly and Company), Bristol Myers Squibb, Daiichi Sankyo, Pfizer, Janssen Pharmaceutical, and Novartis; has received consultancy fee from AbbVie, Ono Pharmaceutical, AstraZeneca, Zenyaku, Kyowa Kirin, Merck Sharp & Dohme, Nippon Shinyaku, Genmab, Mitsubishi Tanabe Pharma, Nippon Kayaku, and Bristol Myers Squibb. K.K. has received honorarium from Ono Pharmaceutical, Eisai, Astellas Pharma, Novartis, Chugai Pharmaceutical, AstraZeneca, Sumitomo Pharma, Kyowa Kirin, Janssen Pharmaceutical, Takeda Pharmaceutical, Otsuka Pharmaceutical, SymBio Pharmaceuticals, Bristol Myers Squibb, Pfizer, Nippon Shinyaku, Daiichi Sankyo, Alexion Pharmaceuticals, AbbVie, Meiji Seika Pharma, Sanofi, Sysmex, Mundipharma, Incyte Corporation, and Kyorin Pharmaceutical; has received research funding from Asahi Kasei Pharma, Eisai, Otsuka Pharmaceutical, Ono Pharmaceutical, Kyowa Kirin, Shionogi, Takeda Pharmaceutical, Sumitomo Dainippon Pharma, Chugai Pharmaceutical, Teijin Pharma, Japan Blood Products Organization, Mochida Pharmaceutical, JCR Pharmaceuticals, Nippon Shinyaku, Chordia Therapeutics, and Meiji Seika Pharma; holds individual stocks in Asahi Genomics; has a patent for Genetic alterations as a biomarker in T-cell lymphomas licensed to Kyoto University and PD-L1 abnormalities as a predictive biomarker for immune checkpoint blockade therapy licensed to Kyoto University; is an Associate Editor of Cancer Science. The other authors declare no potential conflicts of interest.