PD-L1⁺ macrophages are associated with favorable features in primary mediastinal (thymic) large B-cell lymphoma

Abstract

Primary mediastinal (thymic) large B-cell lymphoma (PMBCL) is a rare, aggressive subtype of non-Hodgkin lymphoma and has a complex inflammatory microenvironment. Although most patients can be cured with standard-of-care immunochemotherapy, patients who have disease relapse have an unfavorable prognosis. Pre-treatment prognostic biomarkers in PMBCL are needed. In this retrospective study, we analyzed the clinical features and outcomes of PMBCL patients and their association with immune cell subpopulations identified by multiplex immunofluorescence at initial diagnosis. Two different antibody panels were used to assess macrophages in tissue biopsy specimens collected before the initiation of induction therapy. Twelve PMBCL patients, including five patients who had disease relapse, were included in the analysis. At a median follow-up time of 32.2 months, the median progression-free and overall survival durations were not reached. Our findings suggest that a high density of PD-L1⁺ macrophages is associated with favorable features, such as early disease stage and the absence of B-symptoms, and indicate that a high percentage of PD-L1⁺ macrophages and high densities of CD30⁺PD-L1⁺ cells and CD30⁺ cells might be associated with a lower risk of relapse within 12 months of therapy initiation. Further studies are needed to develop a biomarker signature predictive of treatment response with therapeutic consequences for patients with newly diagnosed PMBCL.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40164-023-00396-0.

To the editor

Primary mediastinal (thymic) large B-cell lymphoma (PMBCL) shares several features with classic Hodgkin lymphoma, such as 9p24.1 amplification, increased programmed cell death protein 1 (PD-1) ligand and CD30 expression [1, 2]. Between 7 and 20% of PMBCL patients have relapse after frontline chemoimmunotherapy, and most have dismal outcomes in spite of intensive therapy [3–5]. Predictive studies are needed to identify PMBCL patients with inferior outcomes, who may benefit from novel therapies or enrollment in a clinical trial.

In this retrospective study, we used multiplex immunofluorescence (mIF) to analyze pretreatment samples from PMBCL patients to identify prognostic characteristics and features associated with treatment response. MIF was not previously used to analyze PMBCL samples and enables better characterization of the cellular composition of the tumor microenvironment in terms of cellular subtypes and has increased sensitivity compared to conventional immunohistochemistry [6]. Methods are described in Additional file 1.

Patient and treatment characteristics are given in Table 1. Twelve PMBCL patients were included in this study. The median age at the time of initial diagnosis was 33 years (range, 22–54 years). Of the 12 patients, 7 (58%) had bulky disease, 5 (42%) had stage I or II disease, 7 (58%) had no B-symptoms, and 8 (67%) had an IPI score of 0 or 1. Nine patients (75%) received 6 cycles of dose-adjusted rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin [4]), as a first line of therapy. However, no patients received consolidative radiotherapy in first complete remission. Five patients had disease relapse at a median of 6 months (range, 3–8 months) and received different additional therapies. At a median follow-up time of 32.2 months (range, 18.3–65.2 months), the median PFS and OS durations were not reached. At the most recent follow-up, one patient had died of progressive disease.

Table 1.

Patient and treatment characteristics

Deidentified number	Gender	Race	Ethnicity	Age at diagnosis	B-SX at DX	Initial stage	IPI	Bulky  > 10 cm	1st line of therapy	At least one relapse	2nd line of therapy	3rd line of therapy	4th line of therapy	FU in months	Status at last FU
PML01	Male	white	not Hispanic	46	No	II	1	Yes	6xDA-R-EPOCH	No				18	Alive
PML02	Female	Asian	not Hispanic	30	No	II	1	Yes	6xDA-R-EPOCH	No				32	Alive
PML03	Male	white	Hispanic	36	No	IV	2	No	6xDA-R-EPOCH	No				21	Alive
PML04	Male	white	not Hispanic	54	No	IV	2	Yes	6xDA-R-EPOCH	Yes	3xR-ICE, ASCT and XRT			65	Alive
PML05	Male	American Indian	not Hispanic	38	No	I	1	Yes	6xDA-R-EPOCH	No				26	Alive
PML06	Female	Hawaiian/Pacific Islander	Hispanic	29	Yes	II	1	Yes	6xDA-R-EPOCH	Yes	1 × R-DHAP	1xR-ICE, Liso-cel		29	Alive
PML07	Male	white	NA	30	Yes	III	1	Yes	1xABVD + 6xDA-R-EPOCH	Yes	3xR-DHAP, then ASCT	Selinexor and Rituximab	Rituximab + fractionated Cyclophosphamide	15	Dead
PML08	Female	white	not Hispanic	24	Yes	IV	2	No	6xDA-R-EPOCH	No				38	Alive
PML09	Female	white	not Hispanic	50	Yes	IV	3	Yes	6xDA-R-EPOCH	Yes	1xR-DHAP	Tisa-cel	7xPembrolizumab + XRT	31	Alive
PML10	Male	white	not Hispanic	22	No	II	0	No	4xDA-R-EPOCH	No				35	Alive
PML11	Male	white	not Hispanic	38	Yes	III	1	No	1xR-CHOP + Len,  + 5xDA-R-EPOCH	No				37	Alive
PML12	Female	white	not Hispanic	28	No	IV	1	No	5xDA-R-EPOCH	Yes	Axi-cel	8xPembrolizumab, ASCT and XRT		57	Alive

ABVD: doxorubicin hydrochloride, bleomycin sulfate, vinblastine sulfate, and dacarbazine; ASCT: autologous stem cell transplantation; axi-cel: axicabtagene ciloleucel; DA-R-EPOCH: dose-adjusted rituximab, etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin; DX: diagnosis; FU: follow-up; IPI: International Prognostic Index; Len: lenalidomide; Liso-cel: lisocabtagene maraleucel; NA: not available; R-DHAP: rituximab, cytosine arabinoside, dexamethasone; R-ICE: rituximab, ifosfamide, carboplatin, and etoposide; SX: symptoms;Tisa-cel: tisagenlecleucel; x: cycles; XRT: radiotherapy of the mediastinum

Per patient, the mean tissue area analyzed was 93.66 mm² (range, 11.6–240.0 mm²) and the mean number of cells analyzed was 9658 cells (range, 6453–12,351 cells). The presented combination of markers output (Additional file 1: Table 1) is based on the previously most commonly assessed combinations [7–9].

Compared with patients who had stage III or IV disease, patients who had stage I or II PMBCL had a significantly higher mean density of CD68⁺PD-L1⁺ cells (115.4 vs. 21.2 cells/mm²; p = 0.02; Fig. 1A, D). Compared with patients with B-symptoms, patients without B-symptoms had a significantly higher mean percentage of CD68⁺PD-L1⁺ cells (26.2% vs. 3.9%; p = 0.01; Fig. 1B). Compared with patients who had disease relapse within 12 months of therapy initiation, patients who did not have relapse within 12 months had a non statistically significant higher mean percentage of CD68⁺PD-L1⁺ macrophages (23.6% vs. 7.6%; p = 0.14; Fig. 1C), higher mean density of CD30⁺PD-L1⁺ cells (156.9 vs. 38.1 cells/mm², p = 0.14; Fig. 1E, Additional file 1: Figure 1), and higher mean density of CD30⁺ cells (2,394.6 vs. 283.5 cells/mm²; p = 0.14; Fig. 1F).

Fig. 1.

A-F Expression of PD-L1 and CD30 by multiplex immunofluorescence on pre-treatment biopsy and their association with clinical characteristics and outcome

Our results suggest that a high density and mean percentage of PD-L1⁺ macrophages in pre-treatment tissue biopsy samples is associated with favorable features, including early-stage disease and the absence of B-symptoms. Moreover, our results indicate that a high percentage of PD-L1⁺ macrophages or high densities of CD30⁺PD-L1⁺ cells or CD30⁺ cells in pre-treatment biopsy samples might be associated with a lower risk of relapse within 12 months of therapy initiation.

The present study had some limitations, including its small, single-center cohort and the lack of markers to precise the phenotype of lymphoma cells and further elements from the immune microenvironment. Further studies with additional patients are necessary to evaluate potential confounding factors such as early-stage disease and the absence of B-symptoms. Besides, additional research is warranted to develop PD-L1⁺ macrophages density and/or percentage as a potential response signature to predict response to checkpoint inhibitors in the frontline setting [10]. We also acknowledge the limitations of our study, including the need for a precise definition of the neoplastic cells since CD30 expression is negative in around 15% of cases, can have a heterogeneous pattern [2] and can be expressed in reactive B-cells of the background. A broader assessment using high-plex technologies, which allows the association of different markers to define specific cell phenotypes, is required to better understand the immune landscape of PMBCL [11]. Several clinical trials of PD-1 inhibitors with or without brentuximab vedotin as frontline therapy for PMBCL (notably NCT04745949 and NCT04759586) are ongoing [12]. The use of targeted therapies in the frontline setting may decrease chemoresistance. However, immune checkpoint inhibitors can cause immune-related adverse events and brentuximab vedotin can induce neurotoxicity and hematotoxicity. Predictive studies to improve the personalization of PMBCL patients and avoid the unnecessary use of certain therapies and their associated risks are an unmet need.

Abbreviations

IPI

International prognosis index scores

mIF

Multiplex immunofluorescence

OS

Overall survival

PFS

Progression-free survival

PMBCL

Primary mediastinal (thymic) large B-cell lymphoma

Author contributions

RES and MLM-P designed the study, analyzed the data, and wrote the manuscript; LF performed the statistical analysis and helped write the manuscript; MN collected data and helped write the manuscript; ERP, FV, JRW, SSN, PS, MRG, CRF, LMS, IIW, SA, RN, and FBH, helped write the manuscript; MLM-P performed multiplex staining. All authors have read and approved the final manuscript.

Funding

This study was supported by the Lymphoma Research Foundation and the Translational Molecular Pathology-Immunoprofiling Laboratory of MD Anderson’s Department of Translational Molecular Pathology.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by the Institutional Review Board of MD Anderson Cancer Center and conducted (protocol 2020–0228) in accordance with our institutional guidelines and the principles of the Declaration of Helsinki. The IRB approved the request of waiver of informed consent and a waiver of authorizations as the study does not involve therapeutic intervention or any type of direct patient contact.

Consent for publication

Not required by IRB.

Competing interests

R.E.S.: Research funding: Rafael Pharmaceuticals, BMS, GSK, and Seattle Genetics. E.R.P. has nothing to disclose. F.V. receives research funding from NCI, CRISP Therapeutics, Allogene, Geron corporation and from the Moonshot program at MDACC. L.F. has nothing to disclose. J.R.W. has received research funding from Kite, BMS, Novartis, Genentech, AstraZeneca, Morphosys/Incyte, ADC Therapeutics, Calithera, Kymera; consulting: Kite, BMS, Novartis, Genentech, AstraZeneca, Morphosys/Incyte, ADC Therapeutics, Calithera, Merck, Abbvie/GenMab, SeaGen, MonteRosa, Iksuda. S.S.N. has received research support from Kite/Gilead, BMS, Cellectis, Poseida, Allogene, Unum Therapeutics, Precision Biosciences, and Adicet Bio; served as Advisory Board Member/Consultant for Kite/Gilead, Merck, Novartis, Sellas Life Sciences, Athenex, Allogene, Incyte, Adicet Bio, BMS, Legend Biotech, Bluebird Bio, Fosun Kite, Sana Biotechnology, Caribou, Astellas Pharma, and Morphosys; has received royalty income from Takeda Pharmaceuticals; has stock options from Longbow Immunotherapy, Inc; and has intellectual property related to cell therapy. P.S. has received research support from Sobi, Astrazeneca Acerta, ADC Therapeutics, ALX Oncology. Advisory board/consultancy: Hutchinson Medipharma, Roche Genentech, Astrazeneca Acerta, ADC Therapeutics, TG Therapeutics, Kite Gilead, Sobi, Incyte Morphosys. M.R.G. reports research funding from Sanofi, Kite/Gilead, Abbvie and Allogene; consulting for Abbvie; honoraria from Tessa Therapeutics, Monte Rosa Therapeutics and Daiichi Sankyo; and stock ownership of KDAc Therapeutics. C.R.F. is consultant for Abbvie, Bayer, BeiGene, Celgene, Denovo Biopharma, Foresight Diagnostics, Genentech/Roche, Genmab, Gilead, Karyopharm, N-Power Medicine, Pharmacyclics/Janssen, SeaGen, Spectrum, has stock Options with Foresight Diagnostics, N-Power Medicine, has research Funding from 4D, Abbvie, Acerta, Adaptimmune, Allogene, Amgen, Bayer, Celgene, Cellectis EMD, Gilead, Genentech/Roche, Guardant, Iovance, Janssen Pharmaceutical, Kite, Morphosys, Nektar,Novartis, Pfizer, Pharmacyclics, Sanofi, Takeda, TG Therapeutics, Xencor, Ziopharm, Burroughs Wellcome Fund, Eastern Cooperative Oncology Group, National Cancer Institute, V Foundation, Cancer Prevention and Research Institute of Texas: CPRIT Scholar in Cancer Research. L.M.S. has nothing to disclose. I.I.W. has provided consulting or advisory roles for AstraZeneca/MedImmune, Bayer, Bristol-Myers Squibb, Genentech/Roche, GlaxoSmithKline, Guardant Health, HTG Molecular Diagnostics, Merck, MSD Oncology, OncoCyte, Jansen, Novartis, Flame Inc, and Pfizer; has received grants and personal fees from Genentech/Roche, Bristol Myers Squibb, AstraZeneca/MedImmune, HTG Molecular, Merck, and Guardant Health; has received personal fees from GlaxoSmithKline and Oncocyte, Daiichi-Sankyo, Roche, Astra Zeneca, Regeneron, Sanofi, Pfizer and Bayer; has received research funding to his institution from 4D Molecular Therapeutics, Adaptimmune, Adaptive Biotechnologies, Akoya Biosciences, Amgen, Bayer, EMD Serono, Genentech, Guardant Health, HTG Molecular Diagnostics, Iovance Biotherapeutics, Johnson & Johnson, Karus Therapeutics, MedImmune, Merck, Novartis, OncoPlex Diagnostics, Pfizer, Takeda, and Novartis. S.A. has received research support paid to her institution from Tessa Therapeutics, SeaGen, Merck, Xencor, Chimagen; Advisory committee member: Sanofi, SeaGen, Tessa Therapeutics; Consultancy: Novartis, Myeloid Therapeutics, Servier, Chimagen. R.N. has received honoraria from Incyte for speaker/preceptorship. F.B.H. has nothing to disclose. M.N. has nothing to disclose. M.L.M-P. has nothing to disclose.