Indolent primary cutaneous B-cell lymphomas resemble persistent antigen reactions without signs of dedifferentiation

Domain: neuroscience
Published: 2026-02-04
Editorial outcome: major_revision

Abstract
Primary cutaneous B-cell lymphomas encompass clinically heterogeneous entities. While primary cutaneous diffuse large B-cell lymphoma, leg type (pcDLBCL-LT) is aggressive, primary cutaneous follicle centre lymphoma (pcFCL) and primary cutaneous marginal zone lymphoma (pcMZL) typically follow an indolent course. To clarify their pathophysiological basis, we perform single-cell RNA sequencing on pcFCL, pcMZL, and pcDLBCL-LT, alongside reactive B-cell rich lymphoid proliferations (rB-LP), gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and systemic counterparts. Here we show that the indolent pcMZL, pcFCL, and rB-LP exhibit a persistent germinal centre reaction, not observed in pcDLBCL-LT or gastric MALT lymphoma. Further, pcMZL top expanded clones develop within lesions from naïve and not post-germinal centre B cells as currently presumed. Our data thus indicate that pcMZL and pcFCL, similar to rB-LP may be driven by (a yet unknown) antigen. While our data indicates that pcFCL exhibits some features of true lymphomas, it clearly supports the classification of pcMZL as a lymphoproliferative disease.

Introduction
Primary cutaneous B-cell lymphomas comprise a heterogeneous group of extranodal non-Hodgkin lymphomas1. The most common subtypes include primary cutaneous marginal zone lymphoma (pcMZL), primary cutaneous follicle centre lymphoma (pcFCL), and primary cutaneous diffuse large B-cell lymphoma, leg type (pcDLBCL-LT)1. pcMZL and pcFCL are generally indolent conditions with a 5-year disease-specific survival of >95%2. In contrast, pcDLBCL-LT is an aggressive disease with 5-year survival rates between 20 and 60%3. For pcMZL, this clinical observation led to a reclassification to a lymphoproliferative disorder in the International Consensus Classification (ICC) joint classification particularly referring to their class-switched cases2. These comprise IgG or IgA positive B-cell proliferations, which follow an indolent clinical course. pcMZL may be preceded by reactive B-cell rich lymphoid proliferations (rB-LP, formerly called “pseudolymphoma”), matching that pcMZL are not veritable lymphomas4. In line, several studies showed that only around 5% of B cells within pcMZL lesions correspond to a top expanded clone5,6. Nevertheless, the WHO classification currently maintains the term lymphoma for pcMZL leading to a conflict between these two classifications7.

Previous gene expression analyses identified characteristic B-cell phenotypes for each primary cutaneous B-cell lymphoma subtype. pcMZL B cells were found to be most similar to plasma cells and pcFCL showed a more germinal centre (GC)-like phenotype, whereas pcDLBCL-LT lesions were most consistent with activated B cells8–10. In cutaneous B-cell lymphomas, clonality was not always detectable using routine molecular methods, or when a surrogate such as light-chain restriction was used11. In those cases, the differentiation from rB-LP can be difficult12. To distinguish pcMZL from rB-LP, skin flow cytometry was found to have superior sensitivity than immunohistochemistry, in situ hybridization or immunoglobulin heavy chain gene rearrangement examinations12. However, its routine use is difficult as it requires fresh tissue, is prone to technical challenges during processing, and is not widely available12. Apart from a more precise molecular characterisation of the disease, the terminology of lymphoma versus lymphoproliferative disorder or cutaneous lymphoid hyperplasia impacts patient education and especially the patients’ perception of their disease13,14.

Here, we present the largest single-cell RNA sequencing characterisation of primary cutaneous B-cell lymphomas and rB-LP and compare these findings to data from their non-cutaneous B-cell counterparts. In this work we show that the indolent pcMZL, pcFCL, and rB-LP exhibit a persistent germinal centre reaction, not observed in pcDLBCL-LT or gastric MALT lymphoma. This indicates that these entities may be driven by (a yet unknown) antigen. Moreover, our data clearly support the classification of pcMZL as a lymphoid proliferation.

Results
scRNA-seq based characterisation matches the current histopathological understanding of primary cutaneous B-cell lymphomas
We obtained fresh biopsies from patients diagnosed with pcMZL (n=9), pcFCL (n=5), pcDLBCL-LT (n=4), or cutaneous rB-LP (n=5), and normal skin from healthy volunteers (NHS, n=4) (Supplementary Table 1). Samples were analysed using single-cell RNA-sequencing (scRNA-seq) coupled with B-cell receptor (BCR) sequencing. After quality control, we obtained data for 268.224 cells (pcDLBCL-LT: 16.908, pcFCL: 71.720, pcMZL: 109.028, rB-LP: 51.336, NHS: 19.232).
Using canonical markers (Supplementary Fig. 1a), we were able to identify T cells and NK cells, B cells, plasma cells, blood vessel and lymphatic endothelial cells, dendritic cells, plasmacytoid dendritic cells, macrophages, melanocytes, keratinocytes, smooth muscle cells, and fibroblasts (Fig. 1a, Supplementary Fig. 1b).
We further subclustered all B cells in order to arrive at a detailed phenotypic characterisation (Fig. 1b). This revealed naïve B cells, as identified by CD19, MS4A1 (CD20), and IGHD, germinal centre (GC) B cells, as identified by CD38, AICDA, and BCL6 that were further subdivided into a light zone (LZ) and dark zone (DZ) GC B-cell population, based on the presence or absence of MKI67 (Fig. 1c). Memory B cells were characterised by their expression of CD19, MS4A1 (CD20), and CD27 with the absence of CD38 and BCL6 (Fig. 1c). Plasma cells were identified through the expression of CD27, CD38, SDC1 (CD138), and the lack of MS4A1 (CD20) (Fig. 1c). Thus, we were able to identify the full spectrum of canonical B-cell phenotypes ranging from naïve B cells to plasma cells.
In addition, we observed aberrant B-cell populations, all originating from pcDLBCL-LT, that formed distinct clusters and could not be classified using canonical B-cell markers (Fig. 1b–e). These were in part MS4A1 (CD20)+, but also expressed MKI67 without the typical expression of CD38 and CD27 for canonical germinal centre B cells (“aberrant B1”, Fig. 1c). Additionally, we observed MS4A1 (CD20) negative B cells that lacked the expression of SDC1 (CD138) and high levels of immunoglobulin associated genes that would be expected for plasma cells (“aberrant B2”, Fig. 1c). This indicates the pcDLBCL-LT contains B cells that no longer match the physiological B-cell phenotypes.
We subsequently performed a differential expression analysis of all of these subtypes (Supplementary Data 1). Matching the phenotypic assignment, naïve B cells primarily showed an up-regulation of activation and signalling associated genes, such as CD69, CD83, and CD44, as well as CXCR4 which is important for chemotaxis and tissue homing. Moreover, they expressed FCER2 (CD21) linked to B cell maturation15. Next to immunoglobulin associated genes, genes highly expressed in plasma cellswere mainly associated with protein synthesis, such as DNAJB9, DNAJC3, PDIA4, and secretion, such as SEC61A1, SEC24D, and SAR1. Germinal centre cells showed a strong expression of cell cycle regulating genes, such as PLK1, CDC20, CCNA2, CCNB1, CCNB2, CDK1, and CDCA3. Finally, the group of aberrant B cells uniquely over-expressed energy metabolism associated genes, such as member of the electron transport chain for ATP production (NDUFA4, NDUFB11, NDUFB4, NDUFB7, NDUFB10, NDUFB9, NDUFV2, NDUFA11, NDUFS6, NDUFS5), but also oxidative phosphorylation (COX5A, COX7B, and COX6A1) and glycolysis (GAPDH, LDHA, LDHB, TPI1, ENO1, PGAM1). This clearly shows that these aberrant B cells were metabolically highly active, matching their proposed malignant phenotype.
To further validate our findings we re-processed publicly available scRNA-seq data from a study on the effect of oncolytic viral therapies on cutaneous B cell lymphomas by Ramelyte et al.16 which contained one pcFCL and one pcDLCBL-LT sample (Fig. 1h, Supplementary Fig. 2). Matching our own data, the pcFCL sample’s clone only consisted of germinal centre B cells (Fig. 1h) while clonally expanded B cells from the pcDLBCL-LT samples showed aberrant phenotypes (Supplementary Fig. 2). These cells were largely CD27 positive with large portions expressing MKI67. Yet, the expected expression of CD38 was lacking. Similarly, naïve or plasma cells were only found in the polyclonal B cell fraction. Therefore, this data fully matches our own observations.

pcMZL originates from pre-germinal centre naïve B cells and follows the canonical B-cell differentiation trajectory
Through our BCR sequencing data, we were able to clearly attribute B cells to the top expanded clone in each disease (Fig. 1d). In each sample, we were thus able to unambiguously distinguish the clonally expanded (presumed malignant) B cells from the polyclonal bystander infiltrate (Supplementary Data 2).
Matching current classifications17, pcMZL and rB-LP uniquely contained relevant numbers of plasma cells (Fig. 1e). Conversely, pcFCL samples showed a dominance of LZ GC B cells (Fig. 1e). pcDLBCL-LT uniquely contained aberrant B-cell phenotypes and lacked relevant numbers of naïve B and canonical plasma cells (Fig. 1e). Our data is therefore in-line with the current histopathological understanding with distinct phenotypic compositions of the B-cell infiltrate for each entity.
pcMZL top clones are considered to derive from post GC B cells17,18. However, we found that clonally expanded B cells from our pcMZL samples spanned all canonical peripheral B-cell phenotypes, from naïve B cells up to terminally differentiated plasma cells (Fig. 1e). We used monocle3 to perform a pseudotime analysis of this data, which matched the canonical B cell differentiation (Fig. 1f, Supplementary Fig. 3). An analysis of the clonally expanded B cells of each pcMZL sample highlighted that the clone seemed to differentiate in each individual sample (Fig. 1g). This was consistent regardless of whether the sample exhibited a predominance of plasma cells or germinal centre B cells and was not limited to specific subtypes of pcMZL as previously described18. This suggests that expanded pcMZL clones develop from naïve, pre-GC B cells irrespective of the phenotypic composition.

Multiplex immunohistochemistry-based characterisation validates scRNA-seq derived composition of B cell infiltrates
To corroborate our scRNA-seq findings, we used a multiplex immunohistochemistry (IHC) approach to simultaneously characterize naïve-like B cells (CD19+/CD20+, CD27−, CD38−, CD138−, CD5−), GC-like B cells (CD19+/CD20+, CD27−, CD38+, CD138−, CD5−), memory-like B cells (CD19+/CD20+, CD27+, CD38−, CD138−, CD5−), and plasma cells (CD19+, C20−, CD138+, CD5−) in a total of 40 samples (Fig. 2a). Matching our scRNA-seq data, pcMZL and rB-LP samples harboured a diverse lymphocytic infiltrate with the highest number of plasma cells (Wilcoxon rank-sum test, FDR corrected p < 0.01), when compared to pcFCL and pcDLBCL-LT (Fig. 2b, c). By contrast, pcFCL samples showed significantly more germinal centre B cells than all other entities (Wilcoxon rank-sum test, FDR corrected p < 0.01). While most pcDLCBL-LT samples were dominated by naïve B cells, some additionally contained high numbers of memory B cells. This directly confirmed our scRNA-seq data as IHC 26 was acquired from the same patient as the scRNA-seq samples patient_117 and patient_207 where we also observed high numbers of CD27+ (aberrant) B cells, similar to the samples from Ramelyte et al.16 (Supplementary Fig. 2). A Shannon diversity index based analysis subsequently confirmed a significantly larger heterogeneity of lymphocyte composition in rB-LP, pcMZL, and pcFCL samples compared to pcDLBCL-LT (Wilcoxon rank-sum test, FDR corrected p < 0.01, D). Overall, these data corroborate our scRNA-seq based characterisation and shows the observed distributions seem stable across larger sample numbers.

Primary cutaneous lymphomas show distinct cellular compositions from their systemic counterparts
It is yet unclear whether our observed phenotypic composition is typical of all B cell lymphomas or specific to CBCL. We thus acquired biopsies from 4 patients diagnosed with gastric mucosa associated lymphoid tissue (MALT) lymphomas refractory to H. pylori eradication (Supplementary Table 1), since this is one of the most frequent marginal zone lymphomas19, and has long been considered the gastric correlate of cutaneous MZL20. Additionally, we integrated public data from studies characterising 18 samples from patients diagnosed with sFCL21, 3 samples of reactive lymph nodes (RLT)21 and 4 samples of sDLBCL22 (Fig. 3a). This resulted in an integrated dataset of a total of 207,634 B cells. Based on canonical markers and differentially expressed genes, we identified all B cells observed in our cutaneous samples, as well as ITGAX+, age associated B cells (ABC)23 (Fig. 3b, Supplementary Data 3).
The expanded clone in gastric MALT lymphoma samples (MALT) was uniquely dominated by CD27+ memory B cells. These showed a class-switch to either IgG or IgA (Fig. 3e), and low numbers of plasma cells (Fig. 3d). B cells of MALT distinctively expressed the genes CD1C and CD1D (Fig. 3f, Supplementary Data 4), which are relevant to lipid antigen processing24. Overall, MALT exhibited a significantly different phenotypic composition compared to pcMZL (Fig. 3g).
pcFCL was the only entity where more than 95% of the clonally expanded B cells were limited to germinal centre B cells (Fig. 3d). In sFCL, the heterogeneity between samples was considerably larger with several sFCL samples also containing clonally expanded memory or plasma cells (Fig. 3d). Matching current literature and diagnostic criteria, pcFCL B cells expressed high levels of BCL6 and sFCL B cells BCL2 (Fig. 3f). Moreover, pcFCL showed the highest expression of MALT1, a proposed target for multiple B cell lymphomas. Otherwise, pcFCL exhibited overlaps with pcMZL in terms of differentially expressed genes (Supplementary Data 4). For example, B cells from both entities expressed FCER2 (CD23), linked to B cell activation and antigen presentation25, HMCES, which limits the accumulation of deletions during somatic hypermutation26, and Lymphocyte Antigen 9 (LY9), a member of the SLAM family of immunomodulatory receptors which is associated with activation and differentiation in T cells27 (Fig. 3f, Supplementary Data 4). Genes associated with tumour progression, such as PCBP2 and TCF4, linked to high risk sDLBCL were significantly down-regulated in indolent CBCL28 (Fig. 3f). Overall, indolent CBCL expressed genes associated with inflammation and immune cell differentiation, while lacking classical tumour promoters.

pcMZL shows significantly lower rates of clonal expansion than other cutaneous and systemic lymphomas
Clonal expansion is considered a hallmark of lymphomas and can be tracked in B cells through BCR sequencing29. We therefore used the BCR data to compare the rate of clonal expansion between cutaneous and systemic lymphomas (Fig. 3h). In pcMZL and rB-LP, clonally expanded B cells represented a median of 4% and 2%, respectively, of all B cells, matching previous reports5. Outliers observed were likely due to sequencing artefacts (Supplementary Fig. 4). This was in contrast to pcFCL and pcDLCBL-LT which showed median clonal expansions of 52% and 97%, respectively. Similarly, in gastric MALT lymphoma the expanded clone accounted for a median of 77% of the total B-cell infiltrate. Moreover, sFCL and sDLBCL were similarly dominated by a single expanded clone, with a median expansion of 96% and 85% respectively. To test the heterogeneity between body sites we acquired two samples simultaneously from a patient with rB-LP (Supplementary Fig. 5). This analysis showed that the estimated clonal expansion was comparable with 6% and 9% between both sites (Supplementary Fig. 5). Additionally, we performed separate analysis of consecutively acquired samples within 6 and 12 months from a patient with rB-LP and pcDLBCL-LT respectively (Supplementary Fig. 6). In both cases, consecutive samples showed matching results with respect to phenotypic composition and rates of clonal expansion (Supplementary Fig. 6). This underscores both the robustness of our method and the temporal consistency of the diseases. Overall, the low rate of clonal expansion in pcMZL substantiate the theory that this condition is more similar to a lymphoproliferative disorder than a true lymphoma.

DLBCL is characterised by aberrant B cell phenotypes that are not found in other entities
Clonally expanded B cells in pcDLBCL-LT could not be annotated following the canonical B-cell development in our samples (Figs. 1c and 3b, c). To arrive at a more detailed characterisation, we performed a subclustering of only pcDLBCL-LT B cells (Supplementary Fig. 7). We observed a loss of MS4A1 (CD20) without the expression of SDC1 (CD138) or CD38 as expected of CD20- plasma cells (Supplementary Fig. 7). There were two clusters of proliferating (MKI67+) B cells. While one matched DZ GC B cells, the other was MS4A1 (CD20)-, with no expression of CD38 or CD27 required to define a germinal centre B cell (Fig. 3b, d, aberrant B2). These findings were consistent with the publicly available scRNA-seq data from Ramelyte et al.16 (Supplementary Fig. 2). This highlight that the presence of aberrant B cell phenotypes is typical of pcDLCBL-LT.
Similar to pcDLCBL-LT, sDLBCL also showed clonally expanded aberrant B cells. This matched the results of a differential expression analysis where sDLBCL and pcDLBCL-LT B cells showed similarities compared to all other entities (Supplementary Data 4). These included the transcription factors and potential oncogenes ATF5 and IRF4, the tumour suppressor RASSF630, the known DLBCL marker IGF2BP331, metabolism and protein synthesis associated genes such as FBP1, GRHPR, SDF2L1, FKBP11, and MZB1, genes protecting against oxidative stress such as PRDX4, and genes associated with survival signals such as CD63 (Fig. 3i). At the same time, FCRL1 which regulates BCR signalling was uniquely lost in pcDLCBL-LT and sDLBCL B cells (Fig. 3i). Simultaneously, the analysis also revealed differences between sDLBCL and pcDLBCL-LT matching their separate clusters. sDLBCL B cells uniquely expressed the potential leukaemia oncogene PARM132, the known ABC-type DLBCL marker MYD88, the B cell proliferation associated marker MYC33, and the tumour suppressor CDKN2C34 (Fig. 3i). pcDLCBL-LT B cells exclusively lacked the poorly characterised gene SMIM14, but also Lymphotoxin Beta (LTB), which is crucial for the development of lymphoid tissues35. This highlights that pcDLBCL-LT is distinct from the indolent CBCL and more similar to its systemic counterpart.

A subset of non-class switched pcMZL samples also showed aberrant phenotypes, as opposed to class-switched pcMZL
Several authors currently differentiate between a class switched and non-class switched variant of pcMZL, where only the non-class switched variant is considered a true lymphoma2,17. In our series, one sample (patient 92) had a clear IgM+, but, surprisingly, also an IgE+ phenotype among the clonally expanded B cells (Fig. 3e). As only IgM and IgG are routinely assessed, this sample would therefore be classified as non-class switched pcMZL in conventional pathological assessments2. Surprisingly, this population contained aberrant B cells (Fig. 3d). Similar to B cells found in pcDLBCL-LT, these cells were MS4A1 (CD20)−, CD27+, IGHM+, with a MKI67+ subpart that showed low expression of CD38 and AICDA but no relevant expression of IGHG1-4- (Supplementary Fig. 8). There were no B cells in this sample that expressed CD138 (SDC1), which would be expected for CD20- B cells (Supplementary Fig. 8). While many aspects of these cells, such as proliferation and potential somatic hypermutation (expression of AICDA) is reminiscent of germinal centre B cells, the lack of CD20 is incompatible with this phenotype, matching reports of CD20 loss in systemic B cell lymphoma36. Clinically, patient 92 has shown an indolent course with two cutaneous recurrences and no sign of systemic disease within a total follow up of 12 years. Therefore, our data suggest that this sample’s clone does primarily consist of aberrant, non-canonical B cells yet with no apparent influence on this patient’s clinical course.
A second sample, WB3, also showed a non-class switched clone (Fig. 3e). Nevertheless, these clonally expanded cells matched canonical germinal centre B cells (Supplementary Fig. 8). In this sample, plasma cells were all polyclonal and IgG4 positive (Supplementary Fig. 8). In a routine histopathological assessment, sample WB3 would therefore be classified as a class-switched pcMZL, as plasma cells would likely have been deemed the pathogenic cell population. This observation highlights that in this specific sample, the histopathological differentiation of class switched vs. non-class switched pcMZL is likely not yet precise enough to distinguish clinically relevant subtypes.

Indolent B-cell lymphomas and rB-LP show similar interactions resembling inflammatory reactions
We performed an interaction analysis using CellChat to further characterise the mechanistic background of CBCL subtypes (Fig. 4a, Supplementary Data 5). All entities showed an interaction between CXCL12 on BEC and FB, APP on most cells of the tumour microenvironment (TME), and THBS1 on BEC which recruit B cells via CXCR4, CD7437, and CD47 respectively38. MIF, a further key chemoattractant via CXCR4 in inflammatory reactions37, was uniquely missing in pcDLBCL-LT. Only pcMZL B cells interacted with PODXL and CD34 on BEC, FB, and LEC via SELL, which allow cell migration39. All B cells expressed IL16 which recruits other immune cells via CD4. Certain interactions were only found in indolent CBCL. For example, B cells interacted with each other through PTPRC and CD22, which regulates the threshold for lymphocyte activation and thereby controls inflammatory reactions40. Similarly, the interaction between FCER2A (CD23) which is key to B cell differentiation41 and ITGAX on DC and MAC was only present on indolent subtypes. Finally, pcDLBCL-LT B cells uniquely interacted with CD27 on B, T, and MEL through CD70 which act as co-stimulatory molecules. Overall, this data matches the current hypothesis that pcDLBCL-LT B cells originate from antigen experienced B cells, while rB-LP, pcMZL, and pcFCL show similar interaction patterns that resemble inflammatory reactions.

Indolent B-cell lymphomas and rB-LP are characterised by ongoing germinal centre reactions
The germinal centre reaction, which consists of a mechanism of ongoing somatic hypermutation, is a key part of B-cell function. We used IgBLAST42 to quantify the rate of somatic hypermutation in all cutaneous samples (Fig. 4b). In all diseases, the top expanded clone showed a higher rate of somatic hypermutation compared to the polyclonal B cells. In pcMZL and rB-LP, the most expanded clone had the widest range of mutations. This matches the assumption that these B cells entered the germinal centre reaction and subsequently acquired mutations to improve antigen specificity, which is in line with the results of our pseudotime analysis of pcMZL samples (Fig. 1g). pcFCL similarly showed increasing rates of somatic hypermutation, yet starting at a considerably higher level (Fig. 4b). This matches our observation that the clone in pcFCL did not contain any naïve B cells (Fig. 1e). Yet, there is evidence of an ongoing acquisition of mutations. Clonally expanded B cells in pcDLCBL-LT showed the highest rates of somatic hypermutation but spanning a much smaller range (Fig. 4b). Despite our characterization of germinal centre B cells in pcDLBCL-LT samples, these data suggest that these cells are largely no longer acquiring new mutations typically associated with the physiological germinal centre reaction. This finding further supports the aberrant phenotypes observed in these samples. In MALT lymphoma samples, polyclonal B cells showed a similar rate of somatic hypermutation as the polyclonal B cells in pcMZL and pcFCL. Yet, the top expanded clone centred around a single value of somatic hypermutations. This matches our phenotypic data where clonally expanded B cells in MALT samples primarily consisted of post-germinal memory B and plasma cells that no longer undergo the germinal centre reaction (Fig. 2e, f). This data suggests that the three indolent cutaneous B-cell reactions, rB-LP, pcMZL, and pcFCL uniquely retain an intact germinal centre reaction.
The germinal centre reaction depends on several other cell types, most importantly CXCR5+ T follicular helper cells (Tfh) and follicular dendritic cells (fDC)43. We therefore subclustered the T cells from all cutaneous samples (Fig. 4c). We were able to identify CD8+, PRF1+, IFNG+ Tc1 polarised cells (CD8 Tc1), some of which were LAG3+ as a sign of exhaustion (CD8 Tc1 exh, Fig. 4d). Further, we observed CD4+, CCR7+, SELL+ CD4 central memory T cells (Tcm), FOXP3+, IL2RA+ regulatory T cells (Treg), MKI67+ proliferating T cells (prolT), PDCD1+, CTLA4+ exhausted CD4+ T cells (CD4 Th2 exh), as well as CXCR5+ T follicular helper cells (Tfh). Tfh were only present in rB-LP, pcMZL, and pcFCL samples (Fig. 4e), matching previous reports where Tfh were histologically observed in all cases of pcMZL44.
Matching their mesenchymal origin with an assumed fibroblast-like progenitor45, fDC are part of fibroblast clusters in scRNA-seq data. We therefore further subclustered all fibroblasts from the cutaneous samples and identified APCDD1+, COL18A1+ secretory papillary (spFB), CCN5+, SLPI+, DPP4+ secretory reticular (srFB), PARD3B+, FBXL7+, AUTS2+ presumably migratory (migFB), COL11A1+, MMP11+, POSTN+ mesenchymal (mesFB), SFRP4+ myo (myoFB), APOE+ CCL19+ inflammatory (iFB) fibroblasts, CR2+, FCER2+, CR1+ CXCL13+ follicular dendritic cells (fDC) and S100B+ SOX10+ Schwann cells (SC) (Fig. 4f, g). Similarly to Tfh, fDC were only observed in rB-LP, pcMZL, and pcFCL samples (Fig. 4h). Therefore, with the detection of an ongoing somatic hypermutation and the presence of Tfh and fDC, samples of rB-LP, pcMZL, and pcFCL uniquely contained all required aspects of a functional germinal centre reaction (Fig. 5).

Methods
Patient recruitment and sample processing
Patients were recruited at the Medical University of Vienna, Austria. Skin punch biopsies or biopsies during gastroscopy were taken after obtaining written informed consent, under a protocol approved by the Ethics Committee of the Medical University of Vienna, which includes sharing of patient characteristics (EK 1360/2018). As per the regulations of the Ethics Committee, patients were only compensated for additional time required for the study participation. Histopathological diagnoses were confirmed by two independent board-certified histopathologists. All patients with pcMZL, pcFCL, and pcDLBCL-LT were further staged using full body CT scans to rule out systemic disease. For all patients with rB-LP, borrelia infection was ruled out. After processing using the Skin Dissociation Kit by Miltenyi Biotech (Bergisch Gladbach, Germany)67–69, single-cell suspensions were subjected to scRNA-seq using the Chromium Single Cell Controller and Single Cell 5’ Library & Gel Bead Kit v2 (10X Genomics, Pleasanton, CA) according to the manufacturer’s protocol. B cell receptor (BCR) sequences were enriched from cDNA using the VDJ Kit workflow by 10X Genomics. Sequencing was performed using the Illumina NovaSeq platform and the 150 bp paired-end configuration.

Data analysis
Raw data from scRNA-seq was preprocessed using CellRanger version 7.0.1 invoking the command “multi” and aligned to the human reference genome assembly “refdata-gex-GRCh38-2020-A” and CellRanger V(D)J segment reference “refdata-cellranger-vdj-GRCh38-alts-ensembl-7.0.0”.
IgBLAST42 v1.20 was used on the BCR filtered contigs to quantify the somatic hypermutation (SHM), presented as ”1-identity in variable region”.
B cell clones were identified from the BCR data through the following steps. Cells featuring more than two contigs or multiple contigs for the same BCR chain (heavy or light chain), and clones with more than two CDR3 chains, were removed. Subsequently, if one of the two most frequent clones only contained one heavy or one light chain were merged with clones were both chains were identified if all cells shared the same V, C, and J genes as the evaluated clonotype, but only if all cells displayed at least 85% sequence identity in both light and heavy CDR3 sequences compared to the reference sequence of the evaluated clonotype, using global pairwise alignment from Biostrings70 R package v2.68 (BLOSUM100 matrix with a gap opening and gap extension penalty values of 10 and 4, respectively).
Every sample was processed using R (version 4.1.0) and Seurat (version 5.0.1)71. Ambient RNA was removed using DecontX72 and doublets removed using scDblFinder73. During the quality assessment, only cells with at least 500 genes and a maximum of 10%, 5% and 1% of mitochondrial, haemoglobin and platelet genes, respectively, were kept for downstream analysis.
Afterwards, samples were normalised and scaled regressing for percentage of mitochondrial genes and sample origin. 30 dimensions were used when invoking the Seurat functions RunPCA, FindNeighbors and RunUMAP. Subsequently, samples were integrated using the FastMNN method74. Leiden algorithm was applied on the shared nearest neighbour graph clustering prior to cell annotation as suggested by Heumos et al.75. In order to achieve a deeper annotation, B and T cells were subclustered, variance was stabilised using SCTransform v276 and ScaleData, respectively, with 15 and 20 dimensions, respectively, for subclusterings. Cell interaction analysis was performed using CellChat v2.277. Differential expression analysis using Seurat’s FindMarkers function with default parameters. Genes were filtered based on FDR (≤0.01) and an expression of 20% within the cells of the respective group (pct.1 and pct.2 for up- and down-regulated genes, respectively).
All p-values reported in the manuscript were corrected for multiple testing using the Benjamini–Hochberg (“FDR”) correction.

Immunohistochemistry
Multiplex immunostainings were conducted as previously described78. Briefly, 4 μm sections were deparaffinized and antigen retrieval was performed in heated citrate buffer (pH 6.0) and/or Tris-EDTA buffer (pH 9) for 30 min. Thereafter, sections were fixed with 7.5% neutralised formaldehyde (SAV Liquid Production GmbH, Flintsbach am Inn, Germany). Each section was subjected to 6 successive rounds of antibody staining, consisting of protein blocking with 20% normal goat serum (Dako, Glostrup, Denmark) in PBS, incubation with primary antibodies, biotinylated anti-mouse/rabbit secondary antibodies and Streptavidin-HRP (Dako), followed by TSA visualisation with fluorophores Opal 520, Opal 540, Opal 570, Opal 620, Opal 650, and Opal 690 (PerkinElmer, Waltham, MA, USA) diluted in 1X Plus Amplification Diluent (PerkinElmer), Ab-TSA complex-stripping in heated citrate buffer (pH 6.0) and/or Tris-EDTA buffer (pH 9) for 30 min, and fixation with 7.5% neutralised formaldehyde. Thereafter, nuclei were counterstained with DAPI (PerkinElmer), and sections were mounted with PermaFluor fluorescence mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). Multiplexed slides were scanned on a Vectra Multispectral Imaging System version 2 following the manufacturer’s protocol (InForm 2.4, PerkinElmer). All phenotyping and subsequent quantifications were performed blinded to the sample identity using QuPath version 0.5.179.

Statistics and reproducibility
All data presented in this manuscript was generated from primary patient samples. Due to limited sample availability technical replicates were not performed. Throughout all analyses, all available samples were used. For scRNA-seq, these were 9 pcMZL, 5 pcFCL, 4 pcDLBCL-LT, 5 cutaneous rB-LP, and 4 samples from healthy volunteers. For IHC analyses, these included 4 rB-LP, 16 pcMZL, 13 pcFCL, and 7 pcDLCBL-LT samples.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study presents a highly detailed single-cell RNA sequencing (scRNA-seq) analysis of primary cutaneous B-cell lymphomas (CBCLs), specifically pcMZL, pcFCL, and pcDLBCL-LT. It highlights new insights into their clonal evolution, transcriptomic profiles, and B-cell receptor (BCR) characteristics. The major claim that pcMZL behaves more like a lymphoproliferative disorder rather than a true lymphoma is potentially paradigm-shifting and offers novel perspectives on diagnostics, classification, and therapeutic considerations.\nKey strengths that justify publication if revised appropriately:\nNovelty & Importance – This study fills a gap in molecular profiling of CBCLs, a rare lymphoma subtype.\nMethodological Rigor – The use of scRNA-seq, BCR sequencing, and multiplex immunohistochemistry (IHC) provides a comprehensive multi-modal analysis.\nWell-Structured & Logical Flow – The manuscript is well-organized, with a clear hypothesis and stepwise data presentation.\nPotential Clinical Impact – The reclassification of pcMZL as a lymphoproliferative disorder could influence future lymphoma classification and management.\nThe study provides strong transcriptomic insights but lacks a direct comparison to existing diagnostic standards. The clinical consequences of pcMZL reclassification need to be clarified.\nProvide a comparison of scRNA-seq to current diagnostics (Page 3, Lines 20-25): compare scRNA-seq vs. IgH rearrangement PCR vs. IHC vs. flow cytometry in terms of diagnostic accuracy; include specificity and sensitivity values where possible.\nPlease, discuss how scRNA-seq findings align with WHO and ICC lymphoma classification criteria and clarify how findings will impact patient management (Page 16, Lines 10-15):\n1. Would patients previously diagnosed with pcMZL now be managed differently?\n2. Should routine scRNA-seq be considered for diagnosis or prognosis?\nCan you make therapeutic implications (Page 21, Lines 5-10) by discussing whether pcMZL patients with high clonal expansion might require targeted therapies or more aggressive treatment?\nIt would be interesting to explore potential immunotherapy targets identified from transcriptomic data.\nShowing clinical applicability will be beneficial and will make findings more translational and relevant to Nature Communications readers.\nIt is a common problem with rare diseases to work with the limited patient cohort (pcMZL n=9, pcFCL n=5, pcDLBCL-LT n=4, rB-LP n=5), the common issue which limits generalizability. In this particular study, the findings are not tested in an independent validation cohort and IHC validation (n=19) is not large enough for meaningful conclusions.\nIt is recommended to expand validation using publicly available datasets (Page 7, Lines 4-10):\nValidate scRNA-seq signatures against GEO datasets (e.g., GSE218861, EGAS00001006052, EGAS00001004904) and to perform bulk RNA-seq validation on an additional independent cohort.\nAdditional technical validation (Page 10, Lines 3-6): the investigators can use spatial transcriptomics to confirm scRNA-seq findings at the tissue level. In addition, multiplex flow cytometry can validate protein expression of key markers in fresh patient samples.\nRegarding statistical robustness (Page 14, Figure 4A), the investigators should consider applying Bonferroni or Benjamini-Hochberg corrections to prevent false positives in differential expression analysis and to use hierarchical mixed-effects models to account for patient-to-patient variability.\nAddressing these concerns would making findings more robust.\nI would recommend improving figures and supplementary data integration including clarifying the labels and legends, improving references the supplementary figures in the main text, and overall improved data presentation (it is not always intuitive for a non-specialist). For example, I would recommend to improve clarity in key figures (Page 8, Figure 2A): add axis labels, clustering annotations, and improved color contrast; provide UMAP embeddings with more detailed explanations.\nPlease, make sure that all supplementary figures are directly linked to main text (Page 6, Figure 1) by explicitly referencing which supplementary figures support key claims. Also, ensure figure legends provide enough information to be understandable on their own. It would be helpful to add summary diagrams (Page 12, Lines 18-22), such as a schematic of B-cell evolution in pcMZL vs. rB-LP would help non-specialists grasp findings more easily. Enhancing readability, accessibility, and figure integration, will make results easier to interpret.\nIt is important to acknowledge study limitations and to justify cohort constraints. Currently, the study does not adequately acknowledge limitations of sample size, demographic bias, and antigen-driven hypotheses. For example, the antigen-driven persistence model is not directly tested, so that suggesting future multi-center studies to validate across diverse cohorts is warranted. To address antigen-driven persistence limitations (Page 15, Lines 8-12), functional validation studies to confirm antigen-driven B-cell expansion should be considered. Furthermore, peptide screening assays to identify putative antigens involved in pcMZL would be of great interest, though might be beyond the scope of this study. Also, longitudinal studies tracking B-cell evolution in pcMZL patients over time might be considered as future directions (Page 24, Conclusion).", "reviewer_2": "This paper tackles the interesting challenge that we have in hematopathology on how to distinguish reactive lymphoid lesions harboring clonal populations from truly neoplastic lesion, and when one should use the term \"lymphoma.\" There are several lymphoproliferative disorders that have clonal B cell populations, but are clearly not malignant, and we, as a field struggle with how to deal with these. This is certainly an important question and one of clinical impact.\nGriss et al ambitiously tackle this problem using single cell sequencing of several of these relatively rare lesions,and report some interesting findings and make some valid arguments about whether particular lesions may be reactive or neoplastic on the basis of the dominance of the main clone, whether the lesion contains \"aberrant cells\" and whether there was persistence of germinal enter reaction. The authors illustrate that our routine approaches in pathology to distinguish lymphoma or not are quite flawed. This was best illustrated by the two examples of the switched and non-switched pcMZL where the authors demonstrate that this criteria is not sufficient to distinguish a lymphoma from a reactive process.\nWhile the manuscript and the data in it were quite interesting, the findings remain mostly descriptive, and are not sufficient to make the broad definitive conclusions that the authors make about these disease entities. There are a number of weaknesses along these lines:\n1. Given the amount of heterogeneity present in these lesions, the number of samples analyzed for each entity is very small. In addition, the authors do not describe how tumors are sampled, and there is no consideration regarding sampling variation, which would impact the % of a clonal population present and the % of supportive cells present, for example. It would be nice to see a few examples of multiple sampling from one tumor to assess the level of variability that may be present just in sampling different areas.\n2. Theses are challenging entities to diagnosis and nomenclature has changed over time. There is no description of how the diagnoses were verified, pathology review, and validation on how the patients were clinically staged. There is limited clinical data , survival data, or consideration of age/gender, etc. This is critical information needed if one will make definitive statements about particular entities.\n3. I think that the way different B cell populations are defined are limited and use minimal markers. For example, aberrant populations seem to be mostly defined by having variation from the normal expression of 2-3 markers.\n4. Independent validation of expression data was quite limited\n5. Assessment of data QC seemed quite limited\nFor all of these reasons, the data, while interesting are not, in my opinion, sufficient to support the broad claims made about pcMZL, pcFL, etc.", "reviewer_3": "The authors have generated and analyzed one of the largest single cell RNA-Seq datasets generated for cutaneous B cell lymphomas. These include pcMZL n=9, pcFCL n=5, pcDLBCL-LT n=4, rB-LP n=5. They do standard informatic analyses to cluster the cell types, to identify the putative clone, and to elucidate the putative cell of origin.\nThe field of cutaneous B cell lymphomas is a burgeoning field with new insights that can have broad implications for 1) cutaneous lymphomas, 2) cutaneous immunology, and 3) B cell biology. They share some markers about the phenotypic diversity in pcMZL that contrasts with the more uniform distribution of the cells in the other cutaneous lymphomas.\nmajor points:\nThe conclusions from this dataset are limited, which I will discuss here.\n1. One of the major questions in the field is how cutaneous and systemic lymphomas are different? Why are the cutaneous B cell lymphomas so indolent? The description of the differences between the systemic and cutaneous lymphomas is inconsistent. A fundamental feature of cutaneous lymphomas is that they are genetically and proteomically distinct from the systemic lymphomas. How much of this can be attributed to the differences in the cell of origin? Restricting much of the analyses to skin lymphomas and skin controls limits the impact. Are the pcDLBCL-LTs a different cluster because the appropriate cells of origin, ABC-type cutaneous lymphomas, are not represented in this dataset? or because they are truly different?\n2. Phenotypic features that distinguish cutaneous and systemic lymphomas are almost completely ignored. Many of these are derived from genetic differences between the cutaneous and systemic counterparts. These fundamental biological insights are not addressed. For example, pcFCL have significantly less BCL2 expression than their systemic counterparts. These comparisons are required to make the analyses interesting.\n3. The use of cutaneous controls is unusual. While there are potential lymphoid aggregates in the skin; traditionally, many of these germinal zone aggregates are formed in the lymph node. It would be better to compare to see if the cells here are similar to the ones found in lymph nodes or different. If so, how?\n4. There is almost no discussion about the microenvironment re: signals between pro-tumor and anti-tumor cells with the lymphoma cells. A wealth of data suggest that many mutations in these CBCLs occur in cell surface proteins that are predicted to mediate interactions with the environment, such as TNFRSF14, FAS, and/or MHC class I. These are not addressed.\n5. These tumors are rare, meaning that it is difficult to expand the dataset to ensure generalizability. In that setting, the manuscript will require orthogonal validation through spatial and/or surface IHC to describe how these markers can be leveraged across larger datasets.\nMinor.\n1. The manuscript is written in a way that makes it difficult to read for non-experts int he field. Tables that describe the differences between clinical entities and their systemic counterparts would make it easier to read.\n2. Genetic information can be gleaned from well curated datasets. Are any mutations observed in these tumors?\n3. The choice of controls such as MALT lymphomas are hard to understand compared to other systemic lymphomas.\n4. Description of the \"bystander\" B cells would be useful. Are these cells defined by merely the absence of the clonal BCR? if so, could this be a technical error? do they express alternative BCRs?"}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "See attached file", "reviewer_2": "The study tackles the challenging area of how to distinguish indolent primary cutaneous lesions and how we should define malignant lymphomas in this setting. The authors have done this through detailed single-cell RNA sequencing analysis of these primary cutaneous lymphomas. Though the paper as originally submitted was definitely intriguing, this author identified a number of weaknesses , including the small n size for the conclusions being made, no independent validation, description of how these cases were classified histologically, limited phenotypic characterization, description QC metrics . The authors have since added new and informative samples and phenotypic and other analyses that address the concerns raised previously. The paper is strong and provides very intriguing evidence that these indolent primary lymphomas are locally driven and provide a basis for future discussion regarding how these should be classified (probably not as malignant lymphomas). However, I have conceptual concern that I believe should be addressed:\nMuch of the evidence that pcMZL and pcFCL may be a locally driven process (and perhaps not truly represent malignant lymphoma) rests in the fact that pcMZL, pcFCL exhibit persistent germinal center reaction, not observed in pcDLBCL and gastric MALT. This represents very strong evidence that pcMZL (especially with the relatively small clonal B populations seen in pcMZL) represents a localized lymphoproliferative disorder and not a true lymphoma.\nHowever, for pcFCL where you have shown larger relative clonal populations, it seems as if there is not sufficient evidence to make the same statement. I wonder about the comparison of pcFL to sFL. FL is a systemic disease and considered malignant, but it also maintains ongoing somatic hypermutation, and also harbors the important elements of the microenvironment, on which these cells are thought to depend. Unlike pcMZL, your data shows that pcFL has the second highest level of ongoing SH with a narrow range (similar to pcDLBCL). It also seems to have a higher clonal percentage. Therefore, the distinction between sFL and pcFL on the parameters that you are using to reclassify pcMZL is less clear.\nI think this should be addressed in the discussion by either 1) providing more evidence or further explaining the distinction between pcFCL and sFL in terms of ongoing SH, clonal populations, microenvironment - or restricting the main hypothesis about the biology of primary cutaneous lymphomas to pcMZLs, and acknowledging that pcFL may represent a neoplastic process whose anatomic restriction remains less understood.\nTo this end, I would suggest modifying the title to focus on \"pcMZL\" and not generally primary cutaneous lymphomas.\nOverall, this is an excellent thought-provoking manuscript that provides new data to help us better understand these primary cutaneous lesions.", "reviewer_3": "The authors present a minimally revised manuscript.\nMy concerns have not been adequately addressed. I will again list them here. in sum, the inability to attempt these analyses suggest a broad ignorance of the field, the underlying B cell lymphoma biology, the techniques accessible to them, and the statistical rigor that additional analyses would provide. The points outlined below are not intended to be complete but rather to highlight the overconfidence by which the authors tried to address scientific points raised by myself and the other reviewers.\n1. The fields of B cell biology already capture parallels between normal B cell biology and B cell lymphomas. This observation does not make their paper impactful. In fact, these descriptions and analogies are baked into their disease entities. The follicular, marginal zone, and diffuse B cell lymphomas have many clear analogies to B cells in different phases of differentiation and expansion. These cells of origin are, in fact, even more detailed as the diffuse large B cell lymphomas comprise multiple clinically relevant phenotypic differences between activated B cell type and germinal cell B cell type. Moreover, there are additional differences amongst the ABC-type leading to clinically relevant differences between nodal and extra nodal where these can be broken down to systemic ABC-type DLBCL, CNS/test ABC-type DLBCL+/- skin, and mediastinal DLBCL. These have now been distributed across additional subtypes based on genetics and transcriptional programs. They have clear differences that can be identified across disease subtypes and across cells-of-origin.\nI and the other reviewers had suggested a careful and thoughtful consideration of these differences to highlight what is truly novel biology unlocked by their single cell RNA-Sequence analyses. These are inadequately covered by the new Figure 3. This should have been thoughtfully done with known markers across disease subtypes and in fact belies an insufficient understanding of the field.\n2. The differences from tumor to tumor and across subtypes strongly correlate with genetics, suggesting a determinative relationship between mutations and phenotypes. Cancers are subject to both copy number and point mutations. There are known mechanisms to identify putative copy number mutations and/or point mutations across single cell RNA-seq datasets. While the authors may not have initially intended to analyze these, their disregard for this suggestion suggests that they under appreciate the importance of genetics to lymphoma biology, which makes it difficult to appreciate whether they understand the field at all and/or the bioinformatics tools that they are deploying.\n.\nFor example, again, there are canonical DLBCL mutations in systemic lymphomas, in skin lymphomas, and shared across the two. There are similar mutations in the other cutaneous B cell lymphoma subtypes.\n3. I have not reviewed or read a lymphoma manuscript with single cell RNA-Seq that has not described the host response to the tumor. CellChat does not describe the tumor microenvironments. It does not describe the cellular composition of the lymphomas across patients and within individual patients.\nThe tumors themselves have genetically-encoded differences in immune ligands. these are not addressed in a systematic way. Their de-prioritization based on CellChat does not mean that they have been actively down regulated by the tumor and/or if this has effects on other cells in the microenvironment. The decision not to pursue this suggests an inadequate appreciation for what tumor immunology represents.\n4. The generalizability of these findings. These studies are the smallest genomic-clinical cohorts that I have seen. The studies with bulk RNA or DNA-Seq have much higher samples which enable broader generalizations of their data. Since these are rare tumors, we had suggested spatial transcriptomics to confirm orthogonally these findings, identify novel spatial interactions, and enable extension of their work to new samples for which fresh single cells are not available. Their claims that this would limit the ability to track antigen-responsive cells (Especially for matched tumors) reflects an ignorance about the potential of integrating these datasets. Again, this highlights an overconfidence in their ability to make conclusions based on their limited understanding of the lymphoma biology, the technical approaches, and the statistical rigor."}, "author_response": "Manuscript number: NCOMMS-24-82894A\nDear Editors,\nThank you very much for your invitation to resubmit our manuscript. We would like to thank all\nthe reviewers for their comments, which we believe have significantly helped to improve the\nmanuscript. Please find below our point-by-point responses to those comments, and we hope\nthat these additions and corrections to the manuscript now warrant publication in your journal.\nReviewer #1 (Remarks to the Author):\nThis study presents a highly detailed single-cell RNA sequencing (scRNA-seq) analysis of primary\ncutaneous B-cell lymphomas (CBCLs), specifically pcMZL, pcFCL, and pcDLBCL-LT. It highlights\nnew insights into their clonal evolution, transcriptomic profiles, and B-cell receptor (BCR)\ncharacteristics. The major claim that pcMZL behaves more like a lymphoproliferative disorder\nrather than a true lymphoma is potentially paradigm-shifting and offers novel perspectives on\ndiagnostics, classification, and therapeutic considerations.\nKey strengths that justify publication if revised appropriately:\nNovelty & Importance – This study fills a gap in molecular profiling of CBCLs, a rare lymphoma\nsubtype.\nMethodological Rigor – The use of scRNA-seq, BCR sequencing, and multiplex\nimmunohistochemistry (IHC) provides a comprehensive multi-modal analysis.\nWell-Structured & Logical Flow – The manuscript is well-organized, with a clear hypothesis and\nstepwise data presentation.\nPotential Clinical Impact – The reclassification of pcMZL as a lymphoproliferative disorder could\ninfluence future lymphoma classification and management.\nThe study provides strong transcriptomic insights but lacks a direct comparison to existing\ndiagnostic standards. The clinical consequences of pcMZL reclassification need to be clarified.\nProvide a comparison of scRNA-seq to current diagnostics (Page 3, Lines 20-25): compare\nscRNA-seq vs. IgH rearrangement PCR vs. IHC vs. flow cytometry in terms of diagnostic accuracy;\ninclude specificity and sensitivity values where possible.\nResponse: We very much thank the reviewer for these suggestions!\nAs suggested, we added an in-depth comparison between cutaneous and systemic lymphoma.\nThese also showed the currently used difference in BCL6 and BCL2 expression between pcFCL and\nsFCL.\nWe additionally did try to perform paraffin-based BCR sequencing using Thermo Fischer’s IonTorrent\nsystem. This is the diagnostic gold-standard in our hospital. Yet, out of the 12 processed samples,\ndespite sufficient DNA, 8 failed to unclear technical issues. Out of the 4, two were deemed as\npolyclonal by the IonTorrent system, while the scRNA-seq data showed a clonal expansion of 50%.\nBased on these results the Department of Molecular Pathology now stopped the use of the\nIonTorrent system for paraffin-based analyses and is working with the manufacturer to analyse\nthese issues.\nIn contrast to cutaneous T cell lymphoma, there is currently no gold standard to diagnose CBCL\nusing flow cytometry.\nFinally, our scRNA-seq sample set is too small to perform robust sensitivity and specificity analyses.\nWe are currently working on different clinically usable assays that could be used on larger sample\nsets and subsequently be fit for routine clinical use. Nevertheless, these efforts are beyond the\nscope of this study.\nPlease, discuss how scRNA-seq findings align with WHO and ICC lymphoma classification criteria\nand clarify how findings will impact patient management (Page 16, Lines 10-15):\n1. Would patients previously diagnosed with pcMZL now be managed differently?\nAs a first consequence patients with non-class switched pcMZL are no longer staged using CT\nscans at our hospital. We hope that our data will help to settle the current discussion on whether\nclass-switched cases of pcMZL are a true lymphoma or not.\nFurthermore, we very much hope and believe that our data will nevertheless spark a wider\ndiscussion into which type of treatments are warranted for the indolent types of CBCL.\nAccordingly, we very strengthened these points in the discussion as follows:\nWe therefore, for the first time, present molecular evidence that aligns with the concept that pcMZL is a\nlymphoproliferative disorder and not a true lymphoma.\n2. Should routine scRNA-seq be considered for diagnosis or prognosis?\nWe believe that we were able to show that scRNA-seq can unambiguously differentiate between\nthese entities. It would therefore definitely be advantageous to at least use such kind of data as a\ngold standard for studies to circumvent current diagnostic challenges.\nWe highlighted this in our discussion as follows:\nOur data thus holds the promise that scRNA-seq of cutaneous samples may be sufficient to distinguish\nbetween primary cutaneous and systemic subtypes of B cell lymphomas arising in the skin.\nCan you make therapeutic implications (Page 21, Lines 5-10) by discussing whether pcMZL\npatients with high clonal expansion might require targeted therapies or more aggressive\ntreatment?\nWe are yet unable to make this statement based on our cohort as we did not have any patient with\npcMZL that showed a high level of clonal expansion. Nevertheless, we strongly believe that we need\nto characterise patients with non-class switched pcMZL more thoroughly. Our data shows that the\nclass-switch alone does not sufficiently identify patients with aggressive diseases.\nIn our dataset, only samples from pcDLBCL-LT showed a clonal expansion similar to systemic\nlymphomas. These patients are already receiving aggressive therapies.\nWe nevertheless do believe that our data offers an avenue for new treatment approaches, as\nhighlighted below.\nIt would be interesting to explore potential immunotherapy targets identified from transcriptomic\ndata. Showing clinical applicability will be beneficial and will make findings more translational and\nrelevant to Nature Communications readers.\nOur data fully supports the current notion that indolent CBCL do not require aggressive treatment.\nWe believe that our data primarily suggest that treatment strategies should focus on identifying the\nunderlying antigen and potentially eliminate it to interrupt the ongoing germinal center reaction.\nWe explicitly added this to our discussion with a new paragraph:\nCurrent treatment strategies for indolent CBCL are primarily targeting the B cells directly. The fact that\nour data strongly suggests that these are antigen-driven reactions offers an avenue for new therapeutic\napproaches. Subsequent studies are needed to functionally validate this finding and identify these\nputatively driving antigens. This could open up options for potentially curative treatment approaches\nthat eliminate the antigen and thereby stop this ongoing reaction.\nIt is a common problem with rare diseases to work with the limited patient cohort (pcMZL n=9,\npcFCL n=5, pcDLBCL-LT n=4, rB-LP n=5), the common issue which limits generalizability. In this\nparticular study, the findings are not tested in an independent validation cohort and IHC validation\n(n=19) is not large enough for meaningful conclusions.\nIn order to strengthen our cohort, we were able to more than double the number of samples used for\nIHC validation (now n = 40). All of these new samples matched the findings from our scRNA-seq\ncohort.\nMoreover, we were able to acquire two new samples from a patient with extensive rB-LP to show\nthat the lesions are similar both with respect to clonal expansion and phenotypic composition.\nFinally, we added a separate analysis of two patients where we acquired samples within 6 and 12\nmonths respectively that also showed that the disease remained unchanged.\nThereby, we believe that we could arrive at a relevant sample size and further support the robustness\nof our methods.\nIt is recommended to expand validation using publicly available datasets (Page 7, Lines 4-10):\nValidate scRNA-seq signatures against GEO datasets (e.g., GSE218861, EGAS00001006052,\nEGAS00001004904) and to perform bulk RNA-seq validation on an additional independent cohort.\nAdditional technical validation (Page 10, Lines 3-6): the investigators can use spatial\ntranscriptomics to confirm scRNA-seq findings at the tissue level. In addition, multiplex flow\ncytometry can validate protein expression of key markers in fresh patient samples.\nAs suggested we added an in-depth analysis and comparison to our data from the only available\nscRNA-seq dataset on primary cutaneous B cell lymphoma (Ramelyte et al., new Figure 1H). These\nsamples fully matched our own analysis.\nAdditionally, we were able to analyse a matched sample from one of our pcDLBCL-LT patients that\nshowed a large number of presumably CD27+ B cells. This was fully confirmed by our IHC analysis\nfurther strengthening the accuracy of our scRNA-seq analysis (new Figure 2).\nRegarding statistical robustness (Page 14, Figure 4A), the investigators should consider applying\nBonferroni or Benjamini-Hochberg corrections to prevent false positives in differential expression\nanalysis and to use hierarchical mixed-effects models to account for patient-to-patient variability.\nAddressing these concerns would making findings more robust.\nAll p-values presented in the manuscript were corrected for multiple testing using the\nBenjamini-Hochberg (\"FDR\") correction. This is now clearly stated throughout the manuscript when\nthe respective p-values are reported. Additionally, we adapted the methods section accordingly:\nAll p-vaues reported in the manuscript were corrected for multiple testing using the\nBenjamini-Hochberg (\"FDR\") correction\nI would recommend improving figures and supplementary data integration including clarifying the\nlabels and legends, improving references the supplementary figures in the main text, and overall\nimproved data presentation (it is not always intuitive for a non-specialist). For example, I would\nrecommend to improve clarity in key figures (Page 8, Figure 2A): add axis labels, clustering\nannotations, and improved color contrast; provide UMAP embeddings with more detailed\nexplanations.\nAs suggested we re-created all supplementary figures and considerably extended the respective\nfigure legends. We very much hope that these are now clearly tied to the main manuscript’s text.\nIn the main manuscript, original figures 2 and 3 were removed and replaced with a new figure\nshowcasing the analysis of our large IHC validation cohort (new Figure 2), as well as the in-depth\ncomparison against systemic lymphoma (new Figure 3). Additionally, we added more graphical\nsummaries to improve the presentation for non-specialists.\nPlease, make sure that all supplementary figures are directly linked to main text (Page 6, Figure 1)\nby explicitly referencing which supplementary figures support key claims. Also, ensure figure\nlegends provide enough information to be understandable on their own. It would be helpful to add\nsummary diagrams (Page 12, Lines 18-22), such as a schematic of B-cell evolution in pcMZL vs.\nrB-LP would help non-specialists grasp findings more easily. Enhancing readability,\naccessibility, and figure integration, will make results easier to interpret.\nAs suggested we adapted all supplementary figures and their legends. Additionally, we added\nthe suggested graphical summary to the new Figure 3.\nIt is important to acknowledge study limitations and to justify cohort constraints. Currently, the\nstudy does not adequately acknowledge limitations of sample size, demographic bias, and\nantigen-driven hypotheses. For example, the antigen-driven persistence model is not directly\ntested, so that suggesting future multi-center studies to validate across diverse cohorts is\nwarranted. To address antigen-driven persistence limitations (Page 15, Lines 8-12), functional\nvalidation studies to confirm antigen-driven B-cell expansion should be considered. Furthermore,\npeptide screening assays to identify putative antigens involved in pcMZL would be of great\ninterest, though might be beyond the scope of this study. Also, longitudinal studies tracking B-cell\nevolution in pcMZL patients over time might be considered as future directions (Page 24,\nConclusion).\nAs suggested, we added a direct comparison of two longitudinal samples from a patient with\nrB-LP and pcDLBCL-LT respectively (new Supplementary Figure 6). These showed that within\nrecurring lesions within 6 and 12 months respectively the level of clonal expansion and\nphenotypic composition did not change.\nNevertheless, we also fully agree that our study can only be a starting point for larger studies\nthat validate our findings. We believe that we provide the strongest evidence yet that indolent\nCBCL may be primarily antigen-driven reactions. Therefore, our data provides a good starting\npoint for such in-depth and larger scale studies. We highlighted this in our discussion:\n[...] Subsequent studies are needed to functionally validate this finding and identify these\nputatively driving antigens. [...]"}, {"round": 3, "reviewer_comments": {"reviewer_2": "As mentioned previously, the data in this paper are interesting and support the idea that pcMZL may be a localized antigen-driven lymphoproliferative disorder as opposed to a true lymphoma. However the evidence in the manuscript that the same may be claimed for pcFL is much weaker in comparison. My suggestion was either to provide more evidence supporting this claim for pcFL – or restricting the claim for primacry cutaneous MZL both I the discussion in the title.\nThe authors’ response is insufficient and presents the data in the manuscript and hypotheses based on this data - in a vacuum , totally ignoring what is known in the field about cutaneous and systemic lymphomas:\n“In contrast to nodal FCL the support cells required for SH are physiologically not present in skin\".\n- It is actually well known that many of the microenvironemntal cells relevant to pCFL are normally present in the skin, though with different frequency and organization, including CD4+ T cells, CD8+ cytotoxic T cells, dermal dendritic cells, macrophages and stromal cells. While Tfh cells are rare in skin, they can be recruited during inflammation.\n\"A true lymphoma such as pcDLBCL-LT therefore lacks these support cells as it is unable to recruit them (in contrast to nodal DLBCL where these support cells are already present).\"\n- This statement does not make sense. Both pcDLBCL-LT and nodal DLBCL are characterized by sheets of large neoplastic and are independent of microenvironment support cells (for the most part). Both PC DLBCL-LT and nodal DLBCL are both malignant neoplasms due to their genetic alterations and obviously does not need to recruit support cells. The differences between these lymphomas do not lie in difference in ability to recruit support cells, but major differences in genetic alterations specific for each entity. These genetic alterations are very well described.\n\"We therefore feel that our hypothesis that pcFCL originates from an initial antigen-driven response is supported (yet not proven) by our data and a more likely explanation than altered B cells that are able to recruit the complete environment that provides necessary survival signals without any signs of systemic disease.\"\n-- This statement is also problematic.\n1) It is still not clear what component of the current data supports the idea that pcFCL originates from an initial antigen-driven response. That data is lacking.\n2) Stating that an entity originates from an initial antigen-driven response is very different than saying an entity is an antigen-driven lymphoproliferative process that is not lymphoma. Gastric MALT - which you correctly describe in the manuscript as true lymphoma originates from an antigen-driven response – namely H.pylori. Sometimes, these lymphomas remain responsive the H.Pylori eradication, and sometimes they become independent of H.pylori stimulation as a result genetic alterations. So whether pcFL initially originates from an antigen-driven response is sort of irrelevant to whether it should be called a lymphoma.\n3) Unfortunately, the authors present their work in a vacuum.\n- There are two current classiifcations in lymphoma – WHO and ICC (International Consensus Classification). Although the WHO5 continues to classificy pcMZL as a lymphoma, the 2022 ICC has already been downgraded it to a persistent antigen-driven “lymphoproliferative disorder” based on current literature, clinical outcomes, association with Borrelia burgdorferi, – so this concept is not new. This should be mentioned in the manuscript. The current data mainly serves to support this point.\n- There is some suggestion in the literature that pcFL also originates from an antigen-driven response , but no great definitive evidence – neither is there in this manuscript. However, there is clear evidence that pcFL exhibits some of the features of a lymphoma – including for examples its tendency to recur locally after excision, and predominance of clonal cells within the lesions. That being said , its not as aggressive as pcDLBCL because it tends to stay in the skin – even when when it recurs, and prognosis is excellent, while pcDLBCL has poor prognosis.\nWe fully share the reviewer’s view that our data is less clear on pcFCL compared to pcMZL. We tried to highlight this in Figure 5 where we tried to clearly show that we do believe that pcFCL cells do acquire some kind of genetic alteration that causes their proliferation.\n- There are papers that demonstrate the phenotypic (BCL2 expression) and genetic alterations (CBP, MLL2, KTM2D, etc) mutations that underlie differences between pcFL and systemic FL that infiltrate skin – this should not be ignored in the manuscript as part of the discussion!!\n\"As it was not our intention to present the evidence for pcFCL at the same level as pcMZL, we adapted our discussion as follows: … Yet, these clones were still undergoing somatic hypermutation as part of the germinal centre reaction, which nevertheless is also observed in sFCL. … … Thereby, the clone may be able to expand, yet still requires the support from the germinal centre environment. As this microenvironment is physiologically not present in skin, we believe that it is most likely that pcFCL develops from a previous antigen-driven reaction. This dependence locks it in its single phenotype and may explain pcFCL’s indolent behaviour. Nevertheless, further studies are needed to determine whether pcFCL should be classified as lymphoma or lymphoproliferative reaction.\"\nTo this reviewer, this explanation is unclear (I am not sure what author is tryng to communicate) and ignores much of what is already in the literature on this topic , as described above\n• PMID: 40227781\n• PMID: 33045225\n• PMID: 33560380\nI would advise authors to really integrate what has already been published in the field, and to focus your conclusions on pcMZL as being not lymphoma - and not pcFL.", "reviewer_3": "The authors have addressed questions satisfactorily"}, "author_response": "Manuscript number: NCOMMS-24-82894A\nDear Editors,\nThank you very much for your invitation to resubmit our manuscript. We would like to thank all\nthe reviewers for their comments, which we believe have significantly helped to improve the\nmanuscript. Please find below our point-by-point responses to those comments, and we hope\nthat these additions and corrections to the manuscript now warrant publication in your journal.\nReviewer #1 (Remarks to the Author):\nThis study presents a highly detailed single-cell RNA sequencing (scRNA-seq) analysis of primary\ncutaneous B-cell lymphomas (CBCLs), specifically pcMZL, pcFCL, and pcDLBCL-LT. It highlights\nnew insights into their clonal evolution, transcriptomic profiles, and B-cell receptor (BCR)\ncharacteristics. The major claim that pcMZL behaves more like a lymphoproliferative disorder\nrather than a true lymphoma is potentially paradigm-shifting and offers novel perspectives on\ndiagnostics, classification, and therapeutic considerations.\nKey strengths that justify publication if revised appropriately:\nNovelty & Importance – This study fills a gap in molecular profiling of CBCLs, a rare lymphoma\nsubtype.\nMethodological Rigor – The use of scRNA-seq, BCR sequencing, and multiplex\nimmunohistochemistry (IHC) provides a comprehensive multi-modal analysis.\nWell-Structured & Logical Flow – The manuscript is well-organized, with a clear hypothesis and\nstepwise data presentation.\nPotential Clinical Impact – The reclassification of pcMZL as a lymphoproliferative disorder could\ninfluence future lymphoma classification and management.\nThe study provides strong transcriptomic insights but lacks a direct comparison to existing\ndiagnostic standards. The clinical consequences of pcMZL reclassification need to be clarified.\nProvide a comparison of scRNA-seq to current diagnostics (Page 3, Lines 20-25): compare\nscRNA-seq vs. IgH rearrangement PCR vs. IHC vs. flow cytometry in terms of diagnostic accuracy;\ninclude specificity and sensitivity values where possible.\nResponse: We very much thank the reviewer for these suggestions!\nAs suggested, we added an in-depth comparison between cutaneous and systemic lymphoma.\nThese also showed the currently used difference in BCL6 and BCL2 expression between pcFCL and\nsFCL.\nWe additionally did try to perform paraffin-based BCR sequencing using Thermo Fischer’s IonTorrent\nsystem. This is the diagnostic gold-standard in our hospital. Yet, out of the 12 processed samples,\ndespite sufficient DNA, 8 failed to unclear technical issues. Out of the 4, two were deemed as\npolyclonal by the IonTorrent system, while the scRNA-seq data showed a clonal expansion of 50%.\nBased on these results the Department of Molecular Pathology now stopped the use of the\nIonTorrent system for paraffin-based analyses and is working with the manufacturer to analyse\nthese issues.\nIn contrast to cutaneous T cell lymphoma, there is currently no gold standard to diagnose CBCL\nusing flow cytometry.\nFinally, our scRNA-seq sample set is too small to perform robust sensitivity and specificity analyses.\nWe are currently working on different clinically usable assays that could be used on larger sample\nsets and subsequently be fit for routine clinical use. Nevertheless, these efforts are beyond the\nscope of this study.\nPlease, discuss how scRNA-seq findings align with WHO and ICC lymphoma classification criteria\nand clarify how findings will impact patient management (Page 16, Lines 10-15):\n1. Would patients previously diagnosed with pcMZL now be managed differently?\nAs a first consequence patients with non-class switched pcMZL are no longer staged using CT\nscans at our hospital. We hope that our data will help to settle the current discussion on whether\nclass-switched cases of pcMZL are a true lymphoma or not.\nFurthermore, we very much hope and believe that our data will nevertheless spark a wider\ndiscussion into which type of treatments are warranted for the indolent types of CBCL.\nAccordingly, we very strengthened these points in the discussion as follows:\nWe therefore, for the first time, present molecular evidence that aligns with the concept that pcMZL is a\nlymphoproliferative disorder and not a true lymphoma.\n2. Should routine scRNA-seq be considered for diagnosis or prognosis?\nWe believe that we were able to show that scRNA-seq can unambiguously differentiate between\nthese entities. It would therefore definitely be advantageous to at least use such kind of data as a\ngold standard for studies to circumvent current diagnostic challenges.\nWe highlighted this in our discussion as follows:\nOur data thus holds the promise that scRNA-seq of cutaneous samples may be sufficient to distinguish\nbetween primary cutaneous and systemic subtypes of B cell lymphomas arising in the skin.\nCan you make therapeutic implications (Page 21, Lines 5-10) by discussing whether pcMZL\npatients with high clonal expansion might require targeted therapies or more aggressive\ntreatment?\nWe are yet unable to make this statement based on our cohort as we did not have any patient with\npcMZL that showed a high level of clonal expansion. Nevertheless, we strongly believe that we need\nto characterise patients with non-class switched pcMZL more thoroughly. Our data shows that the\nclass-switch alone does not sufficiently identify patients with aggressive diseases.\nIn our dataset, only samples from pcDLBCL-LT showed a clonal expansion similar to systemic\nlymphomas. These patients are already receiving aggressive therapies.\nWe nevertheless do believe that our data offers an avenue for new treatment approaches, as\nhighlighted below.\nIt would be interesting to explore potential immunotherapy targets identified from transcriptomic\ndata. Showing clinical applicability will be beneficial and will make findings more translational and\nrelevant to Nature Communications readers.\nOur data fully supports the current notion that indolent CBCL do not require aggressive treatment.\nWe believe that we were able to show that scRNA-seq can unambiguously differentiate between\nthese entities. It would therefore definitely be advantageous to at least use such kind of data as a\ngold standard for studies to circumvent current diagnostic challenges.\nWe highlighted this in our discussion as follows:\nOur data thus holds the promise that scRNA-seq of cutaneous samples may be sufficient to distinguish\nbetween primary cutaneous and systemic subtypes of B cell lymphomas arising in the skin.\nCan you make therapeutic implications (Page 21, Lines 5-10) by discussing whether pcMZL\npatients with high clonal expansion might require targeted therapies or more aggressive\ntreatment?\nWe are yet unable to make this statement based on our cohort as we did not have any patient with\npcMZL that showed a high level of clonal expansion. Nevertheless, we strongly believe that we need\nto characterise patients with non-class switched pcMZL more thoroughly. Our data shows that the\nclass-switch alone does not sufficiently identify patients with aggressive diseases.\nIn our dataset, only samples from pcDLBCL-LT showed a clonal expansion similar to systemic\nlymphomas. These patients are already receiving aggressive therapies.\nWe nevertheless do believe that our data offers an avenue for new treatment approaches, as\nhighlighted below.\nIt would be interesting to explore potential immunotherapy targets identified from transcriptomic\ndata. Showing clinical applicability will be beneficial and will make findings more translational and\nrelevant to Nature Communications readers.\nOur data fully supports the current notion that indolent CBCL do not require aggressive treatment.\nWe believe that our data primarily suggest that treatment strategies should focus on identifying the\nunderlying antigen and potentially eliminate it to interrupt the ongoing germinal center reaction.\nWe explicitly added this to our discussion with a new paragraph:\nCurrent treatment strategies for indolent CBCL are primarily targeting the B cells directly. The fact that\nour data strongly suggests that these are antigen-driven reactions offers an avenue for new therapeutic\napproaches. Subsequent studies are needed to functionally validate this finding and identify these\nputatively driving antigens. This could open up options for potentially curative treatment approaches\nthat eliminate the antigen and thereby stop this ongoing reaction.\nIt is a common problem with rare diseases to work with the limited patient cohort (pcMZL n=9,\npcFCL n=5, pcDLBCL-LT n=4, rB-LP n=5), the common issue which limits generalizability. In this\nparticular study, the findings are not tested in an independent validation cohort and IHC validation\n(n=19) is not large enough for meaningful conclusions.\nIn order to strengthen our cohort, we were able to more than double the number of samples used for\nIHC validation (now n = 40). All of these new samples matched the findings from our scRNA-seq\ncohort.\nMoreover, we were able to acquire two new samples from a patient with extensive rB-LP to show\nthat the lesions are similar both with respect to clonal expansion and phenotypic composition.\nFinally, we added a separate analysis of two patients where we acquired samples within 6 and 12\nmonths respectively that also showed that the disease remained unchanged.\nThereby, we believe that we could arrive at a relevant sample size and further support the robustness\nof our methods.\nIt is recommended to expand validation using publicly available datasets (Page 7, Lines 4-10):\nValidate scRNA-seq signatures against GEO datasets (e.g., GSE218861, EGAS00001006052,\nEGAS00001004904) and to perform bulk RNA-seq validation on an additional independent cohort.\nAdditional technical validation (Page 10, Lines 3-6): the investigators can use spatial\ntranscriptomics to confirm scRNA-seq findings at the tissue level. In addition, multiplex flow\ncytometry can validate protein expression of key markers in fresh patient samples.\nAs suggested we added an in-depth analysis and comparison to our data from the only available\nscRNA-seq dataset on primary cutaneous B cell lymphoma (Ramelyte et al., new Figure 1H). These\nsamples fully matched our own analysis.\nAdditionally, we were able to analyse a matched sample from one of our pcDLBCL-LT patients that\nshowed a large number of presumably CD27+ B cells. This was fully confirmed by our IHC analysis\nfurther strengthening the accuracy of our scRNA-seq analysis (new Figure 2).\nRegarding statistical robustness (Page 14, Figure 4A), the investigators should consider applying\nBonferroni or Benjamini-Hochberg corrections to prevent false positives in differential expression\nanalysis and to use hierarchical mixed-effects models to account for patient-to-patient variability.\nAddressing these concerns would making findings more robust.\nAll p-values presented in the manuscript were corrected for multiple testing using the\nBenjamini-Hochberg (\"FDR\") correction. This is now clearly stated throughout the manuscript when\nthe respective p-values are reported. Additionally, we adapted the methods section accordingly:\nAll p-vaues reported in the manuscript were corrected for multiple testing using the\nBenjamini-Hochberg (\"FDR\") correction\nI would recommend improving figures and supplementary data integration including clarifying the\nlabels and legends, improving references the supplementary figures in the main text, and overall\nimproved data presentation (it is not always intuitive for a non-specialist). For example, I would\nrecommend to improve clarity in key figures (Page 8, Figure 2A): add axis labels, clustering\nannotations, and improved color contrast; provide UMAP embeddings with more detailed\nexplanations.\nAs suggested we re-created all supplementary figures and considerably extended the respective\nfigure legends. We very much hope that these are now clearly tied to the main manuscript’s text.\nIn the main manuscript, original figures 2 and 3 were removed and replaced with a new figure\nshowcasing the analysis of our large IHC validation cohort (new Figure 2), as well as the in-depth\ncomparison against systemic lymphoma (new Figure 3). Additionally, we added more graphical\nsummaries to improve the presentation for non-specialists.\nPlease, make sure that all supplementary figures are directly linked to main text (Page 6, Figure 1)\nby explicitly referencing which supplementary figures support key claims. Also, ensure figure\nlegends provide enough information to be understandable on their own. It would be helpful to add\nsummary diagrams (Page 12, Lines 18-22), such as a schematic of B-cell evolution in pcMZL vs.\nrB-LP would help non-specialists grasp findings more easily. Enhancing readability,\naccessibility, and figure integration, will make results easier to interpret.\nAs suggested we adapted all supplementary figures and their legends. Additionally, we added\nthe suggested graphical summary to the new Figure 3.\nIt is important to acknowledge study limitations and to justify cohort constraints. Currently, the\nstudy does not adequately acknowledge limitations of sample size, demographic bias, and\nantigen-driven hypotheses. For example, the antigen-driven persistence model is not directly\ntested, so that suggesting future multi-center studies to validate across diverse cohorts is\nwarranted. To address antigen-driven persistence limitations (Page 15, Lines 8-12), functional\nvalidation studies to confirm antigen-driven B-cell expansion should be considered. Furthermore,\npeptide screening assays to identify putative antigens involved in pcMZL would be of great\ninterest, though might be beyond the scope of this study. Also, longitudinal studies tracking B-cell\nevolution in pcMZL patients over time might be considered as future directions (Page 24,\nConclusion).\nAs suggested, we added a direct comparison of two longitudinal samples from a patient with\nrB-LP and pcDLBCL-LT respectively (new Supplementary Figure 6). These showed that within\nrecurring lesions within 6 and 12 months respectively the level of clonal expansion and\nphenotypic composition did not change.\nNevertheless, we also fully agree that our study can only be a starting point for larger studies\nthat validate our findings. We believe that we provide the strongest evidence yet that indolent\nCBCL may be primarily antigen-driven reactions. Therefore, our data provides a good starting\npoint for such in-depth and larger scale studies. We highlighted this in our discussion:\n[...] Subsequent studies are needed to functionally validate this finding and identify these\nputatively driving antigens. [...]"}]

Decision letter