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. 2025 Sep 27;9(9):e70200. doi: 10.1002/hem3.70200

Xq24/IL13RA1 aberrations as key drivers of female bias in primary mediastinal large B‐cell lymphoma

Lukas Marcelis 1,2, Ryan Nicolaas Allsop 3,4, Marlies Vanden Bempt 5,6,7,8, Koen Debackere 5,6,7,9, Félicien Renard 5,6,7,8, Stefania Tuveri 7, Daan Dierickx 9, Adrian Janiszewski 3,4, Bradley Philip Balaton 3,4, Johanna Vets 1,2, Barbara Dewaele 7, Lucienne Michaux 7, Peter Vandenberghe 7,9, Jan Cools 6,7, Joris Robert Vermeesch 7, Thomas Tousseyn 1,2, Vincent Pasque 3,4, Iwona Wlodarska 7,✉
PMCID: PMC12475935  PMID: 41017961

Abstract

Female‐biased incidence in primary mediastinal large B‐cell lymphoma (PMBCL) is enigmatic and points to a potential contribution of the X chromosome in this disease. To elucidate the postulated involvement of X‐linked factor(s), we profiled the X chromosome in 48 diagnostic PMBCLs of male (21) and female (27) origin. Molecular cytogenetic analysis detected copy number gain of X/Xq in all male patients and 59.3% (16/27) of female patients. The remaining female cases revealed either a cytogenetically cryptic copy‐neutral loss of heterozygosity (CNLOH) of X/Xq (14.8%) or germline XX (25.9%). Remarkably, RNAseq data of 28 cases indicated a nonrandom involvement of transcriptionally active X homolog in gain/CNLOH, validated by hXIST RNA‐fluorescence in situ hybridization (FISH) in two PMBCL‐derived cell lines. Further transcriptomic analysis revealed IL13RA1 (Xq24) as the target of the Xq aberrations. In agreement with this, the vast majority (32/38, 84.2%) of PMBCL cases were IL13RA1‐positive by immunohistochemistry. The intriguing finding of IL13RA1 protein expression in female PMBCLs with germline XX suggests epigenetic reactivation and expression of IL13RA1 on the inactive X. Functional in vitro studies performed on Ba/F3 cells showed that overexpressed IL13RA1 is potent to transform murine pro‐B cells and constitutively activate the oncogenic JAK‐STAT signaling pathway. The novel pathogenic Xq24/IL13RA1 defects appeared as disease‐defining aberrations driving PMBCL and contributing to the related sex disparity. Our findings indicate that female predominance in PMBCL is due to the higher risk of women of acquiring pathogenic Xq24/IL13RA1 defects by female‐exclusive genetic/epigenetic mechanisms (CN‐LOHX and reactivation of IL13RA1 on Xi) not operating in males.

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INTRODUCTION

Primary mediastinal large B‐cell lymphoma (PMBCL) is a rare aggressive neoplasm predominantly affecting young women. 1 The lymphoma involves the mediastinum and arises from a thymic B cell. 2 The molecular pathogenesis of PMBCL is not yet fully understood, but multiple lines of evidence indicate a prominent involvement of the NFkB and JAK‐STAT signaling pathways, the programmed cell death protein 1 (PD‐1)‐mediated immune evasion, and epigenetic instability pathways. 3 , 4 The components of these pathways are commonly targeted by genetic aberrations. The most frequent abnormalities observed in 40%–81% of PMBCL cases include copy number (CN) gain of 9p24.3 (JAK2‐CD274‐PDCD1LG2), 6p22.2 (HIST1H1C), 2p16.1 (REL), and 9q34.3 (TRAF2), copy‐neutral loss of heterozygosity (CNLOH) of 6p/MHC, and mutations in SOCS1, B2M, ITPKB, STAT6, NFKBIE, and GNA13. 3 , 4 , 5 , 6

Sex disparities observed in PMBCL are very intriguing and enigmatic. While lymphoma, like the majority of human cancers, is more prevalent in males than females, 7 PMBCL affects females twice as often as males. Whether this is due to the X chromosome housing important biological regulators and cancer‐related genes 8 , 9 remains unknown. The baseline genetics of the X chromosome, present in a single copy in males and two copies in females, is more complex than that of autosomes. 10 , 11 To balance X‐linked gene dosage between sexes, cells with two activated X chromosomes (XaXa) undergo a process known as X‐chromosome inactivation (XCI) in early human development, whereby one of the two chromosomes is inactivated (XaXi). 12 , 13 The epigenetic silencing of X is initiated by the X‐inactive specific transcript (XIST), a noncoding RNA gene located at Xq13. 14 Female‐exclusive expression of XIST is a hallmark of Xi, and the Xi can be observed as a distinctive structure in interphase nuclei (Barr body). 15 The chromosome‐wide transcriptional silencing of X, however, is incomplete, and more than 20% of X‐linked genes in females escape XCI (escapees) and remain constitutively or facultatively expressed from the otherwise inactive X homolog. 10 , 14 , 16 , 17 , 18 In particular, female lymphocytes are predisposed to increased expression from the Xi. 19

The commonly observed sex disparities in cancer, particularly the higher cancer risk observed among males, are attracting the growing interest of scientists. 20 , 21 , 22 , 23 , 24 Although molecular mechanisms underlying this phenomenon remain poorly understood, recent studies indicate an important role of X‐linked genes, particularly escapees, in the sex bias in tumorigenesis. 25 , 26 , 27 The pan‐cancer analysis performed by Dunford et al. 25 revealed male‐biased mutations in six so‐called “escape from X‐inactivation tumor suppressors” (EXITS) (ATRX, CNKSR2, DDX3X, KDM5C, KDM6A, and MAGEC3). Given that functional loss of these genes in men may be achieved by a single mutation while their inactivation in females requires two molecular hits, EXITS genes could substantially contribute to the observed higher incidence of these tumors in males. Whether impaired XCI and/or X‐linked genes contribute to sex bias in female cancers, including PMBCL, merits investigation. Unfortunately, the recent molecular genetic studies of PMBCL routinely omitted X chromosome, 3 , 4 , 28 and so far, the question of sex disparity in PMBCL was addressed only by one group. 4 Despite a dedicated X chromosome‐specific genetic analysis, no mechanism underlying this phenomenon was identified.

In this study, we performed a comprehensive analysis of X‐linked aberrations in PMBCL, resulting in the identification of pervasive primary genetic defects of the X chromosome that target IL13RA1/Xq24 and contribute to the female bias in PMBCL.

MATERIAL AND METHODS

Study material

Patients with the histopathological diagnosis of PMBCL were retrospectively selected from the clinical database according to the availability of diagnostic biopsy material, DNA and/or archival cytogenetic pellets, and clinical follow‐up data. Pretreatment specimens were retrieved from the tumor banks of our institution. Constitutional DNA from peripheral blood was available for eight cases. The study cohort comprises 48 cases, including 21 male and 27 female gender. The cases were reviewed by two hematopathologists (L.M. and T.T.) for definite classification in accordance with both the recent World Health Organization (WHO) classification of hematolymphoid tumors 1 and the 2022 International Consensus Classification of Mature Lymphoid Neoplasms. 29 All cases but one had bulky mediastinal disease with a mature B‐cell immunophenotype and at least partial expression of CD23 and/or CD30. In cases with limited CD23 or CD30 expression, the presence of the compartmentalizing fibrosis distinctive for this entity was considered essential. The cases showed frequent gains of 9p24 (86%) and 2p16 (37.2%) by diagnostic molecular cytogenetics. One patient, 25‐year‐old woman without mediastinal involvement, was also included. Her tumor, located in the small intestine, showed typical morphological features, including interstitial fibrosis and strong diffuse expression of CD30, CD23, and MAL by immunohistochemistry (IHC), as well as CN gain of 9p24, similar to bona fide PMBCL. 30

Relevant clinical characteristics of the selected patients are summarized in Table 1. Median age, clinical presentation, outcome, and current status of male and female patients were similar. Most of the male patients, however, presented in Stage II (77.7%), while female patients presented equally (38.5%) in Stage II and Stage IV (data not shown). Eight cases with nodular sclerosis classic Hodgkin lymphoma (NSCHL) were additionally selected for IHC. Two PMBCL‐derived cell lines, Karpas 1106P 31 and U2940, 32 were purchased from European Collection of Authenticated Cell Cultures (ECACC) and American Type Culture Collection (ATCC), respectively. Two NSCHL‐derived cell lines, L428 and L1236, were purchased from DSMZ (German Collection of Microorganisms and Cell Cultures). The Ethics Committee UZ/KUL Leuven approved this retrospective study and renounced the need for written informed consent (S56035).

Table 1.

Clinical characteristics of the primary mediastinal large B‐cell lymphoma cohort.

Clinical characteristics Data
n (%)
Female 27 (56.2)
Male 21 (43.8)
Age at diagnosis Median (range) years
Female 34 (12–76)
Male 33 (14–53)
Stage n (%)
I/II 34 (70.8%)
III/IV 13 (27.1)
NA 1 (2.1)
Mediastinal involvement n (%)
Yes 47 (97.9)
No 1 (2.1)
First‐line treatment n (%)
CHOP 2 (4.2)
R‐CHOP 15 (31.2)
Combined modalitya 24 (50.0)
Other 6 (12.5)
NA 1 (2.1)
Response n (%)
Durable complete 32 (66.7)
Remission after first line 11 (22.9)
Relapsed/refractory disease 4 (8.3)
NA 1 (2.1)
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Abbreviations: CHOP, cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone; NA, not available; R‐CHOP, rituximab‐CHOP.

a

Combination of chemotherapy and radiotherapy.

Methods

Applied methods, including conventional and molecular cytogenetics, RNA‐fluorescence in situ hybridization (FISH), IHC, next‐generation sequencing, and in vitro functional studies are described in Supporting Information S12.

RESULTS

Male PMBCL patients show gains of X/Xq

This project started with the identification of a novel t(X;14)(p11.4;q32.33) associated with a supernumerary der(X) in three male PMBCL (M‐PMBCL) patients (Table 2, Figure 1A). FISH analysis of Case 1 (Supporting Information S1: Table 1) confirmed duplication of der(X), rearrangement of IGH (14q32.33), and excluded involvement of GPR34 (Xp11.4), the gene targeted by the known t(X;14)(p11.4;q32.33) in marginal zone lymphoma 33 (Figure 1B–D). Subsequent FISH analysis with Xp‐specific DNA probes followed by array‐based comparative genomic hybridization (aCGH) analysis performed in Case 3 mapped the Xp11.4 breakpoint telomeric to BCOR (~132 kb) (chrX: 39530321–39893990 bp [hg19]) (Figure 1E,F, Supporting Information S1: Table 1). Given its localization in a gene‐poor region [MID1IPA, the first coding gene on der(14), is ~1.25 Mb away], molecular consequences of the IGH‐driven t(X;14) remain elusive.

Table 2.

Relevant characteristics of male primary mediastinal large B‐cell lymphoma.

Genetic featuresa
Case no. Age Applied techniques X chromosome profile Known gains RNAseq X/A ratio IHC for IL13RA1
t(X;14)‐positive
1 34 CCG, FISH t(X;14)(p11.4;q32), +der(X)t(X;14)(p11.4;q32) 9p24 A NA
2 53 CCG, FISH, aCGH t(X;14)(p11.4;q32), +der(X)t(X;14)(p11.4;q32) 9p24 NA +
3 34 CCG, FISH t(X;14)(p11.4;q32), +der(X)t(X;14)(p11.4;q32) 9p24 NA +
t(X;14)‐negative
4 43 SNPa,b FISH X,+Xq10q28 2p16, 9p24 A +
5 18 SNPa,b FISH X,+X 9p24 A −
6 29 SNPa,b FISH X,+X 9p24 A +
7 45 SNPa,b FISH X,+X 2p16, 9p24 A +
8 14 SNPa,b FISH X,+X 2p16 A +
9 41 SNPa,b FISH X,+X 2p16, 9p24 A NA
10 31 SNPa,b FISH X,+X 9p24 A +
11 30 SNPa,b FISH X,+X 2p16, 9p24 A +
12 44 SNPa,b FISH X,+X 9p24 A −
13 40 UPS X,amp(Xq22.1q27.3) 9p24 NA +
14 25 CCG, FISH X,+X NA NA +
15 32 CCG, FISH X,+X 9p24 NA NA
16 34 CCG, FISH X,+X 9p24 NA NA
17 26 FISH CEPX x2, Xp11.4 ×2 NA NA +
18 29 FISH CEPX x2, Xp11.4 ×2 9p24 NA +
19 23 FISH CEPX x2, Xp11.4 ×2 9p24 NA NA
20 37 FISH CEPX x2, Xp11.4 ×2 NA NA NA
21 30 FISH CEPX x2, Xp11.4 ×2 NA NA NA
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Abbreviations: A, analyzed; aCGH, array‐based comparative genomic hybridization; ccfDNA, circulating cell‐free DNA; CCG, conventional cytogenetics; CEPX, chromosome X centromeric probe; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; M, male; NA, not analyzed; SNPa, single‐nucleotide polymorphism array; UPS, ultralow pass sequencing of ccfDNA; X/A, X‐to‐autosome expression ratio.

a

The reported genetic aberrations were found in 20%–60% of karyotyped metaphase cells and/or 12%–53% of interphase cells analyzed by interphase FISH.

b

Genome‐wide SNPa profiles have been previously described. 6

Figure 1.

Figure 1

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Genetic mapping of t(X;14)(p11.4;q32.33) performed in three cases of male primary mediastinal large B‐cell lymphoma. (A) Partial G‐banded karyotype of Case 1 showing t(X;14)(p11.4;q32.33),+der(X)(p11.4;q32.33). (B–E) Examples of metaphase fluorescence in situ hybridization with whole chromosome painting (WCP) probes for X (red) and 14 (green) (B), LSI IGH BA (C), RP11‐20I14 (green) and RP11‐645P13 (red) (D), and BCOR BA (E). Genomic locations of the probes are shown in Supporting Information S1: Table 1. Red and blue arrows indicate der(14) and both copies of der(X), respectively. (F) Array comparative genomic hybridization profile of the X chromosome defined in Case 3 showing one (normal) copy of Xp11.4p22.3 and gain of Xp11.4 q28. Split signals in (C)‐(D)/(E) prove breakpoints in the region of IGH and distal to BCOR, respectively.

The intriguing duplication of der(X) in all three M‐PMBCL cases with t(X;14)(p11.4;q32.33) prompted us to examine the genetic profile of the X chromosome in 18 well‐documented M‐PMBCL cases (Table 2). These cases were subsequently analyzed by three‐color interphase FISH (iFISH) with centromeric probes (CEPs) for X, Y, and 6 (ploidy level control). The analysis identified cell clones with an extra CEPX signal (X,+X) in all samples (Table 2, examples in Figure 2A,B, right panel). Subsequent single‐nucleotide polymorphism (SNP) array analysis (Cases 4–12) (examples in Figure 2A,B, left panel) and ultralow pass sequencing (UPS) of circulating cell‐free DNA 34 conducted in Case 13 confirmed iFISH data. Notably, Cases 4 and 13 revealed partial duplication of X, limited to Xq and the selectively amplified Xq22.1q27.3 region, respectively.

Figure 2.

Figure 2

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Examples of chromosome X imbalances detected by single‐nucleotide polymorphism (SNP) array (left panel) and fluorescence in situ hybridization (FISH)/cytogenetics (right panel) in two M‐PMBCLs (A,B), four F‐PMBCLs (C–F), and two cell lines (G,H). Red lines in SNP array profiles indicate log ratio 0. Red and green boxes frame gained regions and copy‐neutral loss of heterozygosity (CNLOH) stretches (allele plots), respectively. Probes for interphase FISH (iFISH) performed in Cases 8 (A) and 10 (B) include centromeric probe (CEP)‐X (red)/‐Y (green)/‐6 (blue) (ploidy control) and CEP‐X (green)/‐6 (blue) in Case 44 (D). Case 29 with CNLOH (two Xq) (C) and Case 34 with a partial copy number gain of Xq (two extra copies) (E) were analyzed with Xq24 probes (red) and break apart probes for JAK2 applied to identify neoplastic cells hallmarked by 9p24 gain.

Chromosome X aberrations are common in female PMBCL patients

To determine the frequency of X‐related CN alterations in female patients, in the next step, we performed iFISH (Supporting Information S1: Table 1), SNP array, and/or UPS of circulating cell‐free DNA analysis on a series of 27 diagnostic female PMBCL (F‐PMBCL) cases and two female‐derived PMBCL cell lines (Karpas 1106 P and U2940). Results are summarized in Table 3. Cytogenetic data were available in four cases. In addition to the previously discussed clonal genomic imbalances, 6 the cases showed either a germline XX configuration (plus sporadic local deletions) (Cases 22–28) or a cytogenetically cryptic CNLOH 6 (alias uniparental disomy) of X/Xq (Cases 29–32) or whole/partial CN gain of X (Cases 33–48). Examples of the identified X chromosome aberrations are shown in Figure 2C–F. Interestingly, cytogenetic and FISH analysis of Case 48 identified the neoplastic stemline with a sole gain of X (47,XX,+X) accompanied by secondary cytogenetic aberrations in the coexisting major sideline (Supporting Information S7: Figure 1). SNP array profiling performed on Karpas 1106P and U2940 confirmed the known X chromosome abnormalities 35 , 36 and identified two novel CNLOHX lesions in U2940 (Figure 2H).

Table 3.

Relevant characteristics of female primary mediastinal large B‐cell lymphoma (PMBCL).

Case no. Age Genetic featuresa RNAseq X/A ratio IHC for IL13RA1
Applied techniques X chromosome profile Known gains
1. XX
22 34 SNPa,b FISH XX 2p16 A +
23 35 SNPa,b FISH XX 9p24 A −
24 41 SNPa,b FISH X,del(X)(p22.31p22.2) ‐ A +
25 48 SNPa,b FISH XX 9p24 A −
26 27 SNPa,b FISH XX ‐ NA +
27 20 SNPa,b FISH X, del(X)(q23q26.3)sc 9p24 NA +
28 37 SNPa,b FISH XX 9p24 A +
2. CNLOHX
29 41 SNPab X,CNLOHX 9p24 NA +
30 12 SNPab X,CNLOHX 9p24 A +
31 76 SNPab X,CNLOHX 2p16, 9p24 A +
32 16 SNPab X,CNLOHXp22.33q26.3,+Xq26.3q28 ‐ A +
3. Whole or partial gain of X
33 29 SNPab X,CNLOHXp11.1p22.33, del(X)(q11.2q21.1),+Xq21.1q28 2p16, 9p24 A +
34 40 SNPab X,CNLOHXp22.1p22.33, +Xp22.1q28,+Xq22.1q26.3 2p16, 9p24 NA +
35 25 SNPab XX,+Xp22.33p22.2,+Xp22.2p11.4, +Xp11.4q28 2p16 A +
36 35 CCG, SNPab X,i(Xq),+i(Xq) (del(X)(p11.1p22.33), +Xq11.2q28,+Xq11.2q28,+Xq11.2q28) 9p24 A −
37 45 SNPab X,del(X)(p22.13p22.33), +Xp22.13q28,+Xq21.1q28 2p16, 9p24 A +
38 40 UPS, SNPab XX,+X ‐ A +
39 32 SNPab XX,+X 2p16, 9p24 A +
40 45 SNPab XX,+X 2p16, 9p24 NA +
41 29 SNPab XX,+X 2p16, 9p24 A +
42 37 SNPab XX,+X 9p24 A −
43 32 SNPab XX,+X 2p16, 9p24 A +
44 20 SNPab XX,+X 9p24 A +
45 27 CCG, FISH XX,+X 9p24 NA NA
46 31 CCG, FISH XX,+X 9p24 NA NA
47 30 UPS XX,+X 9p24 NA NA
48 37 CCG, FISH XX,+X(14%)/sl,+t(X;7)(q26;p11)(86%) ‐ NA +
PMBCL‐derived cell lines
49 Karpas 1106P ‐ SNPab XX,+X,+Xq11.1q21.1 2p16, 9p24 NA −
50 U2940 ‐ SNPab X,del(X)(p11.2p22.33), CNLOHXq11.2q13.1, del(X)(q13.1q21.3) hmz, CNLOHXq21.3q28 ‐ NA −
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Abbreviations: A, analyzed; ccfDNA, circulating cell‐free DNA; CCG, conventional cytogenetics; CNLOHX, copy‐neutral loss of heterozygoisity of chromosome X; F, female; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; NA, not analyzed; SNPa, single‐nucleotide polymorphism array; UPS, ultralow pass sequencing of ccfDNA; X/A, X‐to‐autosomes expression ratio.

a

The reported genetic aberrations were found in 25%–47% of karyotyped metaphase cells and/or 15%–62% of interphase cells analyzed by interphase FISH.

b

Genome‐wide SNPa profiles have been previously described. 6

Figure 3A summarizes X chromosome imbalances identified in altogether 37 profiled M‐ and F‐PMBCL cases. Compilation of the data allowed to define the smallest common region of gain/CNLOH. This ~43 Mb region located at Xq21.1q27.3 (chrX: 99500001–14250000 bp [hg19]) represents the amplified Xq segment in Case 13 (Figure 3B). Notably, it covers a smaller Xq22.1q26.3 (chrX: 100890285–137943387 bp [hg19]) area selectively gained in Case 33.

Figure 3.

Figure 3

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Mapping of the smallest region of copy number gain/copy‐neutral loss of heterozygosity (CNLOH) of Xq. (A) Schematic summary of the X chromosome imbalances in 38 profiled primary mediastinal large B‐cell lymphoma (PMBCL) cases. Each bar shows aberrations detected in cases indicated in the upper row. (1) denotes the smallest region of gain/CNLOH and (2) the selectively triplicated region in Case 34. (B) Genome‐wide profile and the zoomed X chromosome profile of Case 13 (male PMBCL) analyzed by ultralow pass sequencing of circulating cell‐free DNA. Blue, green, bordeaux, and red dots denote balanced, lost, gained, and amplified regions, respectively. In the zoomed monosomic X chromosome, green denotes normal regions.

Common gain of an active X chromosome in PMBCL

To determine whether X chromosome imbalances in PMBCL are random (affect both Xa and Xi) or nonrandom (affect only Xa or Xi), we sought to determine the activation status of the involved X chromosome in available PMBCL cases. Twenty‐eight tumor samples (10 M‐PMBCL and 18 F‐PMBCL) and eight control (not malignant) lymph node samples (four of male and four of female origin) were subjected to bulk RNA sequencing. For analysis, the samples were divided into four groups according to sex and X‐linked chromosomal aberrations: (1) M‐PMBCL,+X/Xq (Cases 1, 4–12), (2) F‐PMBCL,+X/Xq (Cases 33, 35–39, and 41–44), (3) F‐PMBCL,CNLOHX (Cases 30–32), and (4) F‐PMBCL,XX (Cases 22–25, 28). The ratio of average X‐linked gene expression to average autosomal gene expression (X‐to‐autosome expression ratio) 37 was used to determine if there was an increase in X‐linked gene expression compared to controls (Supporting Information S2: Table 2). We found significantly increased X‐to‐autosome expression ratio in cases representing M‐PMBCL,+X/Xq and F‐PMBCL,+X/Xq when compared to male and female controls, respectively (Figure 4A). A trend toward the increased ratio is seen in F‐PMBCL,CNLOHX samples, but it is not significant due to low sample size (three samples). In the merged F‐PMBCL,CNLOHX and F‐PMBCL,+X/Xq group (data not shown), the increased ratio is significant (P = 0.000 1029). The X‐to‐autosome expression ratio remained unchanged in F‐PMBCL,XX samples. These findings suggest that the X chromosome, which was gained or affected by CNLOH in M‐ and F‐PMBCL, is active (Xa). In other words, gain of X/CNLOHX in PMBCL is nonrandom and selectively affects Xa.

Figure 4.

Figure 4

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Activated status of X chromosomes in primary mediastinal large B‐cell lymphoma (PMBCL). (A) X/A expression ratio analysis. PMBCL samples were stratified into four groups according to sex and X‐chromosome aberrations. +X/Xq indicates whole or partial gain‐of‐X, XX indicates X‐copy‐neutral samples, and CNLOHX indicates samples with acquired CNLOHX. Boxplots show the X‐to‐autosome expression ratio for samples within each group, as well as for controls (ctrl). The box plots display the median (horizontal black line), the interquartile range (box limits), and the whiskers represent 1.5× of the interquartile range. P‐values were calculated by Student's t‐test. (B) Examples of hXist (488 fluorophore‐labeled) RNA‐fluorescence in situ hybridization in KARPAS P1106 (left column), U2940 (middle column), and female fibroblast cell lines (right column). The signal for hXist is present in female fibroblast, lost in U2940 (CNLOHXq), and present in KARPAS P1106 (gain of X and Xq11.1q21.1). F‐PMBCL, female PMBCL; M‐PMBCL, male PMBCL; DAPI, 4',6‐diamidino‐2‐phenylindole.

To validate the data, RNA‐FISH analysis with hXIST (Xq13.2) and RNA probes for X‐linked genes HDAC8 (Xq13.1) and HUWE1 (Xp11.22) was performed on Karpas 1106P and U2940 cell lines (Figure 4B and Supporting Information S8: Figure 2A). A female human fibroblast cell line was used as a control. The number of signals for each probe was counted per cell in 60 informative cells while blinded for both probes and cell lines (Supporting Information S8: Figure 2B). In female fibroblast cells (XaXi), single hXIST (Xi), hHDAC8 (Xa), and hHUWE1 (Xa) signals were observed. All Karpas 1106P cells showed one hXist signal, and a proportion of cells revealed two hHDAC8 and two hHUWE1 signals indicative of gain of Xa. Lack of the hXIST signal and presence of two hHDAC8 signals and one hHUWE1 signal in a proportion of cells of U2940 remain in line with CNLOH‐associated loss of Xi and a partial duplication of Xa (Table 3 and Figure 2H).

Gain of an active X chromosome in PMBCL is associated with IL13RA1 expression

Given that X‐linked gene expression is increased in the majority of PMBCL samples, we sought to investigate which X‐linked genes within the smallest region of gain/CNLOH (Xq22.1q27.3/99.5‐142.5 Mb) were deregulated in PMBCL cases. Of the 817 protein‐coding genes in this ~43 Mb region, only a minority were dysregulated across the PMBCL subgroups (Figure 5A–F) (Supporting Information S9: Figure 3A). Twenty‐four genes were commonly upregulated in four PMBCL subgroups with +X/CNLOH (Supporting Information S9: Figure 3B, Supporting Information S2: Table 2). These genes included both IL13RA1 and NKRF, which have previously been linked to both immune function and cancer. 38 , 39 , 40 , 41 , 42 , 43 , 44 Based on previous observations of distinctive IL13RA1 expression in PMBCL 45 , 46 and tumor suppressor features of NKRF, 43 , 44 we considered IL13RA1 as the best candidate gene. In our study setup, no significantly expressed X‐genes were found in the F‐PMBCL,XX subgroup (Figure 5E). Both cell lines revealed enhanced expression of the Xq genes, but a majority of them, including IL13RA1, were not significantly upregulated (Figure 5F).

Figure 5.

Figure 5

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Expression analysis of genes located in the smallest region of gain/copy‐neutral loss of heterozygosity (CNLOH) of Xq. Volcano plots show the differential gene expression for (A) male primary mediastinal large B‐cell lymphoma (PMBCL) samples versus male controls (non‐malignant lymph nodes), (B) female X‐copy‐neutral PMBCL samples versus female controls, (C) female (partial) gain‐of‐X PMBCL samples versus female controls, (D) female (partial) CNLOHX/partial gain‐of‐X samples versus female controls, (E) all male and female PMBCL samples versus all male and female controls, and (F) PMBCL cell lines versus female controls. Only genes located within the region Xq22.1q27.3 are shown. P‐values given by the Mann–Whitney U test were converted to q‐values. (G)–(J) IL13RA1 immunohistochemistry. (G) Representative IL13RA1‐positive PMBCL, (H) representative IL13RA1‐negative PMBCL, (I) a non‐neoplastic lymph node germinal center node with staining of germinal B cells, (J) nodular sclerosing Hodgkin lymphoma with staining of the Hodgkin/Reed‐Sternberg cells. All pictures were taken on a Leica DM 2000LED microscope with a DFC 290 HD camera. Scalebar = 50 µm. F‐PMBCL, female PMBCL; M‐PMBCL, male PMBCL.

Common expression of IL13RA1 in PMBCL

The level of IL13RA1 protein expression in PMBCL was examined by IHC. IL13RA1 serum (PA528309, ThermoFisher) was applied in 4 nonmalignant lymph node biopsies, 38 available PMBCL cases, both cell lines, and additionally, in 8 cases of NSCHL (Supporting Information S5: Table 5). In “reactive” lymph nodes, the antibody stained germinal center centroblasts and follicular dendritic cells, like in murine lymph nodes. 47 The majority of PMBCL tumors (13/15 M‐PMBCL and 19/23 F‐PMBCL cases) showed positive immunostaining (Tables 2 and 3), while both cell lines were IL13RA1‐negative. All NSCHL cases revealed a membrane expression of the IL13RA1 protein. Representative examples of IHC staining are shown in Figure 5G–J. Validation of the IHC findings by RNA in situ hybridization for IL‐13Rα1 48 would be advisable.

Frequent mutations in components of the IL4‐IL13 signaling pathway

To investigate protein‐coding alterations in PMBCL, whole‐exome sequencing (WES) analysis was performed in 19 PMBCL cases, including 8 tumor/normal tissue paired cases (nos 5, 8, 10, 11, 22, 25, 42, and 43). Both cell lines were subjected to whole‐genome sequencing (WGS). 6 The cohort comprised 9 M‐PMBCL samples (+X/Xq) and 10 F‐PMBCL samples (5 with XX, 3 with CNLOHX/Xq, and 2 with +X). Mutation data for eight cases (tumor/normal pairs) and the cell lines have already been published. 6 Here, we have focused on the mutation status of the components of the IL4/IL13 signaling pathway (Figure 6A). The results, integrated with relevant SNP array and IL13RA1 IHC data, are shown in Figure 6B. Mutations of SOCS1, STAT6, IL4, and IL13RA1 were detected in 67%, 52%, 43%, and 19% of samples, respectively. Four out of five F‐PMBCL with a germline XX showed mutations in either STAT6 (no. 28) or SOCS1 (no. 22) or IL13RA1 and SOCS1 (no. 26) or SOCS1 and STAT6 (no. 27). The known IL4R/IL13RA1 mutations in Karpas 1106P and U2940 3 were confirmed. The localization of IL13RA1 mutations detected by us and Noerenberg et al. 5 is shown in Figure 6C. All these mutations are localized in a region where amino acid changes are predicted to have a high pathogenicity score. 49 Clonality and hierarchy of the acquired mutations were estimated using the cancer cell fraction (CCF) analysis 5 applied for 56 genes mutated in at least three samples. Cutoff of a CCF of 95% was used to call the mutation clonal. The genes presented in Supporting Information S10: Figure 4A are ordered according to the reduced degree of clonality. Given that the most clonal mutations are considered early genomic events and the most subclonal are considered late genomic events, 5 the hierarchy of acquired mutations in components of the IL4/IL13 pathway is as follows: STAT6 (87.5%), IL4A (73%), SOCS1 (59%), and IL13RA1 (34%). The CCF of individual mutations in the analyzed genes is shown in Supporting Information S11: Figure 4B.

Figure 6.

Figure 6

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Mutation and functional analysis of components of the IL4–IL13 signaling pathway. (A) Schematic overview of the IL13R pathway. (B) Selected mutation data for IL4/IL13 signaling components, relevant single‐nucleotide polymorphism array, and IL13RA1 IHC data. No mutation(s) is marked in gray. (C) Schematic overview of the IL13RA1 protein, with mutations found in primary mediastinal large B‐cell lymphoma indicated. Mutations reported by Noerenberg et al. 5 are shown in gray. The pathogenicity score as calculated by AlphaMissense 49 is shown below. (D) Normalized counts showing expression of IL13 signaling components in Ba/F3 cells. (E) Growth curve showing transformation to IL3‐independent growth of Ba/F3 cells with and without IL13RA1 overexpression (left). On Day 30, IL13 was removed from half of the cells previously supplemented with IL13 (right). (F) GFP% of IL13RA1‐overexpressing Ba/F3 cells after IL3 removal. (G) GFP levels at Day 40 after IL3 removal. (H) Western blot showing expression and/or phosphorylation of Jak1, Jak2, Tyk2, Stat6, Socs1, GFP, and b‐actin in IL3‐dependent Ba/F3 cells (Ctrl) and IL13RA1‐overexpressing Ba/F3 cells (IL13RA1). (I) Dose response curve for Ba/F3 cells with and without IL13RA1 overexpression treated with tofacitinib (JAK3/2/1 inhibitor) for 48 h. (J) Dose response curve for Ba/F3 cells with and without IL13RA1 overexpression treated with ruxolitinib (JAK1/2 inhibitor) for 48 h. (K) IL13RA1 expression levels and CRISPR dependency score for cell lines of hematological malignancies (DepMap). (L) Treatment of L1236 cells with 1 µM ruxolitinib or 1 µM tofacitinib for 48 h. (M) Treatment of L428 cells with 1 µM ruxolitinib or 1 µM tofacitinib for 48 h. DMSO, dimethyl sulfoxide.

IL13RA1 expression confers cytokine‐independent growth

To functionally examine the oncogenic capacities of IL13RA1 overexpression, we used the murine IL3‐dependent pro‐B cell line Ba/F3, a popular model system developed for assessing the transforming potency of kinase oncogenes. 50 Low/undetectable expression levels of endogenous Il13ra1 and Il13ra2 and high expression of Il4ra (Figure 6D), suggesting that the IL13 signaling is not active in Ba/F3 cells, made this model particularly suitable for our study. We cloned the human IL13RA1 ORF in the pMIG vector with an IRES‐GFP reporter. Retroviral transduction of Ba/F3 cells with the vector (but not with an empty vector) led to IL13RA1 overexpression and conferred IL3‐independent growth (Figure 6E). Notably, transformation with IL13RA1 was much faster when the cells were stimulated with IL13. The subsequent depletion of IL13, however, had no effect on the viability of the transformed cells (Figure 6F,G). As shown in Figure 6H, stimulation with IL13 led to a stronger activation of the IL13 signaling pathway, illustrated by a downstream phosphorylation of Stat6. Remarkably, after transformation, we observed a strong selection for cells with low/undetectable Socs1 expression, which is in accordance with a frequent mutation‐driven inactivation of SOCS1 in PMBCL. 3 , 4 , 5 Ba/F3 cells transformed by IL13RA1 overexpression were sensitive to JAK‐STAT inhibition (Figure 6I,J). To validate our findings in a human model, we used the L428 and L1236 Hodgkin Lymphoma cell lines, as these cell lines show high expression levels of IL13RA1. According to public CRISPR dependency data (https://depmap.org), the L1236 cell line is strongly dependent on IL13RA1, while this is much less pronounced for L428 (Figure 6K). In line with this observation, we could show that L1236 cells are sensitive to JAK‐STAT inhibition, while L428 cells are not (Figure 6L,M).

DISCUSSION

PMBCL is one of the rare nonreproductive system tumors preferentially affecting females (F:M/2:1). To gain insight into mechanisms driving sex‐biased incidence in PMBCL and potential X‐linked factors contributing to this phenomenon, we analyzed genetic profiles of the X chromosome in a series of diagnostic male (21) and female (27) PMBCL cases, as recommended. 51 Molecular cytogenetic analysis not only confirmed the early observations of recurrent CN gain of X in this tumor 52 , 53 , 54 , 55 but, for the first time, demonstrated that the frequency of this aneuploidy significantly varies between sexes, being present in 100% of M‐ and ~60% (16/27) of F‐PMBCL. The remaining female patients showed either a cytogenetically cryptic CNLOHX (14.8%) or a germline XX configuration (25.9%) (Figure 7). Of particular importance was the RNAseq‐based discovery of a nonrandom involvement of activated X homolog in CN gain and CNLOH, leading to a significant disruption of the X‐gene dosage compensation in PMBCL. Further analysis identified IL13RA1 (Xq24) as the X‐linked factor potentially targeted in PMBCL. This choice was supported by its genetic localization in the smallest common region of CN gain/CNLOH, significant upregulation at mRNA and protein levels in the vast majority of analyzed cases, and a functional link to both immune function and cancer. 38 , 39 , 40 , 41 , 42 The molecular basis of IL13RA1 expression protein in the enigmatic F‐PMBCL cases with a germline XaXi configuration remains elusive. We hypothesize that IL13RA1 may be escaping XCI in these tumor samples (Figure 7B), resulting in sufficient expression to generate IL13RA1 protein detectable by IHC and to drive disease, but not enough to cause a significant increase in expression in the study setup. Our hypothesis is supported by previous studies of escapees showing their significantly lower expression from the Xi than the Xa. 17 Also, the lack of analogous M‐PMBCL,XY cases expressing IL13RA1 excludes the contribution of unknown Xi‐unrelated mechanisms in this process. Although IL13RA1 is not a constitutive escapee, 56 , 57 , 58 previous reports have already shown that the gene can variably escape XCI in nonmalignant and malignant cells. 15 , 59 , 60 Our RNAseq‐based findings were validated on two PMBCL‐derived cell lines subjected to hXIST RNA‐FISH (Figure 4B). Notably, Karpas 1106P and U2940 showed a low expression level of mRNA and protein. By analogy with the IL7RA‐driven T‐ALL, 61 we hypothesize that the established PMBCL‐derived cell lines may no longer require high levels of IL13RA1, particularly in the presence of IL4R‐driving mutations (Figure 6A). 3 The findings reported here require validation on a larger series of cases. Given, however, the abundance of skipped X‐linked data in PMBCL generated by international groups, this task could be achieved without special experimental efforts.

Figure 7.

Figure 7

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Pathogenic events in primary mediastinal large B‐cell lymphoma (PMBCL). (A) All male patients are featured by copy number gain of activated X/Xq due to mitotic nondisjunction (ND). (B) The mechanism of ND operates in only ~60% of female patients. The remaining female PMBCLs (F‐PMBCLs) acquire an activated X/Xq by copy‐neutral loss of heterozygosity (CNLOH; loss of Xi and duplication of Xa) (~15%) or undergo the IL13RA1‐related escape from X chromosome inactivation (EsXCI), resulting in a biallelic expression of IL13RA1. Notably, latter mechanisms affecting Xa and/or Xi are female‐exclusive. Further selection and clonal proliferation (S/CP) of the aberrant thymic B cells is followed by acquisition and accumulation of numerous secondary mutations (MUT) beneficial for the growth and progression of PMBCL. Red and violet chromosomes represent Xa and Xi, respectively. Black and white boxes at Xq24 denote active and inactive alleles of IL13RA1, respectively, and lightning symbolizes mutational hits. (C) Summary of the pathogenic Xq24‐related events identified in both genders, explaining the phenomenon of female‐biased incidence in PMBCL. Created in BioRender. M‐PMBCL, male PMBCL.

It is worth emphasizing that a distinctive expression of IL13RA1 in PMBCL has already been reported in previously published gene‐expression‐based studies. 45 , 46 Notably, Rosenwald et al. 45 recognized IL13RA1 as the third gene (following PDL2 and SNFT) that best discriminates PMBCL from DLBCL (1.9‐fold change compared with DLBCL 62 ) and the top gene in the PMBCL predictor. To the best of our knowledge, however, molecular mechanisms underlying aberrant expression of IL13RA1 in PMBCL have never been explored. IL13RA1 codes for the IL‐13Rα1 protein that, together with IL4Rα, constitutes the IL4‐receptor type II shared by the T cell‐derived IL4 and IL13. 38 , 39 The cytokines signal through the JAK‐STAT cascade, leading to activation of JAK2, phosphorylation of STAT6, and subsequent promotion of transcription of STAT6‐targeted genes controlling lymphocyte proliferation and activation, and directly affecting tumor proliferation. 40 Although constitutive activation of the JAK‐STAT signaling pathway is well documented in PMBCL, 63 , 64 , 65 the stimulus responsible for this phenomenon has not been elucidated.

Functional studies of IL13RA1 conducted on Ba/F3, a murine IL‐3‐dependent pro‐B cell line, showed that overexpressed IL13RA1 can transform these cells and induce their IL3‐independent growth by activation of the downstream JAK‐STAT cascade. Transforming potential of IL13RA1 was stronger under stimulation with IL13, but once transformed, the cells did not need IL13 supplementation for growth. Importantly, the transformed Ba/F3 cells were sensitive to JAK‐STAT inhibition. The choice of a murine but not human model is a serious limitation of this work, but the demonstration of a transforming potential of IL13RA1 on already transformed/engineered human cells is challenging. Despite these limitations, our study provides new insight into molecular mechanisms underlying the constitutive activation of the oncogenic JAK‐STAT signaling pathway in PMBCL. Until now, this process was exclusively associated with somatic mutations in the involved genes and 9p24 (JAK2/JMJD2C) amplification. 3 , 4 , 64 , 65 Our research indicates that the JAK‐STAT cascade is primarily activated by enhanced expression of the gene‐dosage‐sensitive IL13RA1, due to the gain of its transcriptionally active allele or, likely, by epigenetic reactivation of the silenced allele on Xi. Cytogenetic finding of a sole +X in the stemline of case 48 (Supporting Information S7: Figure 1) proves the pivotal role of this primary genetic aberration in the pathogenesis of PMBCL. Evidently, secondary JAK/STAT‐related mutations, including mutations in IL13RA1 5 (Figure 6B,C), increase signal strength through this pathway and presumably are beneficial for driving the growth of lymphoma cells and disease progression.

It is worth noting that NSCHL is also featured by an enhanced expression of IL13RA1 (Figure 5J). However, in contrast to PMBCL, activation of IL13RA1 and the downstream JAK‐STAT signaling pathway in this tumor is driven by autocrine IL13 production by neoplastic Hodgkin cells. 48 , 65 PMBCL and NSCHL show numerous unique similarities in clinical presentation, including mediastinal involvement and young patients' age at diagnosis, cytogenetic hallmarks (amplification of 9p24 and 2p16), and gene expression profiles. 46 , 48 , 66 , 67 On the other hand, distinct morphologic and immunophenotypic characteristics of both lymphomas 62 indicate their different cell‐of‐origin. According to the latest WHO classification, 1 NSCHL displays a slight female predominance. Lack of hallmarking Xq24 imbalances in NSCHL (unpublished data 68 ), driving aberrant expression of IL13RA1 and female bias in PMBCL, additionally supports the distinction between PMBCL and NSCHL.

Genetic mechanisms contributing to the sex disparity in PMBCL are summarized in Figure 7. The biased F:M/2:1 incidence ratio in this tumor seems to result from a higher risk for females of acquisition of the Xq24/IL13RA1 aberrations. While both genders have a similar risk of gain of Xa by nondisjunction, females may be exposed to additional genetic mechanisms affecting Xa and/or Xi (CNLOH, escape from XCI), not operating in males (Figure 7C). We presume that the remaining nonreproductive system female cancers are driven by similar X‐related mechanisms. Currently available data have already documented frequent genetic and epigenetic aberrations of Xa/Xi in at least three female cancers, including the well‐studied breast carcinoma (BC) 69 , 70 , 71 , 72 , 73 and two rare subtypes of renal cell carcinoma (RCC) (chromophobe RCC 74 and the Xp11 translocation RCC [tRCC] 75 ). In the context of our research, noteworthy is a common gain of X chromosome in male BC 76 and loss of the supernumerary Xi followed by multiplication of Xa in rare male BC diagnosed in patients with Klinefelter syndrome. 77 The important role of X chromosome (enriched for immunity‐related genes) in female‐biased autoimmune disorders has been widely discussed. 78 , 79 , 80

To summarize, our study provides a completely new view on the role of the X chromosome in the pathogenesis of PMBCL. We showed that all PMBCL cases, regardless of gender, are hallmarked by genetic or likely epigenetic Xq24 defects resulting in increased gene dosage/expression of IL13RA1 and constitutive activation of the oncogenic JAK‐STAT signaling pathway. These pathogenic hits appear as primary disease‐defining aberrations and enhanced expression of IL13RA1 as a sine qua non condition for initiation of lymphomagenesis. Complicity of other X‐linked protein‐coding and ‐noncoding factors in this process, however, is not excluded. Importantly, the Xq24 aberrations play a critical role in the sex disparity in PMBCL. Our findings support the hypothesis that female‐biased cancers are driven by overexpressed X‐linked oncogenes, while male‐biased cancers are initiated by inactivating mutations in X‐linked tumor suppressor genes. 11 The findings reported here, extrapolated on other female cancers, including BC, may pave the way for the identification of novel X‐linked oncogenic factors. Finally, our study points to IL13RA1 as a new potential pharmacological target in the treatment of patients with PMBCL. Given the known role of the IL4/13 signaling pathways in inflammatory diseases and cancer, both cytokines and their receptors are attracting growing interest from the scientific community. 81 , 82 Further developments in molecular therapeutic approaches and cell therapies targeting the IL13/IL13RA1 axis are warranted.

AUTHOR CONTRIBUTIONS

Lukas Marcelis: Conceptualization; investigation; writing—original draft; visualization; validation; methodology. Ryan Nicolaas Allsop: Investigation; writing—original draft; writing—review and editing; visualization; validation; formal analysis. Marlies Vanden Bempt: Conceptualization; investigation; writing—original draft; validation; methodology. Koen Debackere: Investigation; writing—original draft; validation; visualization; formal analysis; methodology. Félicien Renard: Investigation; methodology. Stefania Tuveri: Investigation; formal analysis; methodology. Daan Dierickx: Data curation; funding acquisition; supervision; resources. Adrian Janiszewski: Investigation. Bradley Philip Balaton: Writing—original draft; conceptualization. Johanna Vets: Investigation; validation; visualization. Barbara Dewaele: Investigation; data curation; resources. Lucienne Michaux: Investigation; writing—original draft; writing—review and editing; data curation; resources. Peter Vandenberghe: Data curation; funding acquisition; resources. Jan Cools: Writing—original draft; funding acquisition; supervision. Joris Robert Vermeesch: Funding acquisition; supervision; methodology. Thomas Tousseyn: Investigation; supervision; resources; data curation. Vincent Pasque: Conceptualization; investigation; funding acquisition; writing—original draft; supervision; methodology. Iwona Wlodarska: Conceptualization; investigation; writing—original draft; writing—review and editing; visualization; project administration; supervision; data curation.

CONFLICT OF INTEREST STATEMENT

J. C. is an Editor at HemaSphere. The other authors declare no conflicts of interest.

FUNDING

KU Leuven Research Fund (C14/21/119); the FWO and F.R.S.‐FNRS (G0I7822N); Fonds Wetenschappelijk Onderzoek (11L0722N, 1263323N, 1S74420N, G0B4420N, and G0C9320N); and Stichting Tegen Kanker.

Supporting information

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ACKNOWLEDGMENTS

The authors would like to thank Ursula Pluys, Kathleen Doms, Kim Rummens, Julie Vanderheydt, and Anne Renmans for their excellent technical assistance. Dedicated to Chris De Wolf‐Peeters.

DATA AVAILABILITY STATEMENT

SNPa, WGS, and WES data are deposited in GEO (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE184212), ENA (https://www.ebi.ac.uk/ena/browser/view/PRJEB50976), and EGA (EGAS00001006235), respectively.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information.

HEM3-9-e70200-s012.docx (19.1KB, docx)

Supporting Information.

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HEM3-9-e70200-s006.docx (23.6KB, docx)

Data Availability Statement

SNPa, WGS, and WES data are deposited in GEO (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE184212), ENA (https://www.ebi.ac.uk/ena/browser/view/PRJEB50976), and EGA (EGAS00001006235), respectively.

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