Overlapping features of primary cutaneous marginal zone lymphoproliferative disorder and primary cutaneous CD4+ small/medium T cell lymphoproliferative disorder: A diagnostic challenge examined by genomic analysis

Abstract

Primary cutaneous marginal zone lymphoproliferative disorder (PCMZL) and primary cutaneous CD4+ small/medium T cell lymphoproliferative disorder (CD4+ TLPD) are indolent lymphoproliferative disorders. However, cases with overlapping features can be challenging. We identified 56 CD4+ TLPD and 38 PCMZL cases from our pathology archives. Clinical, morphologic and immunophenotypic features were reviewed. PCR for immunoglobulin (IG) and T-cell receptor gamma (TRG) gene rearrangements were analyzed. NGS studies were performed on 26 cases with adequate material, 19 with CD4+ TLPD and 7 with PCMZL. CD4+ TLPD presented mostly (91%) as solitary lesions, located in the head and neck area (64%), while PCMZL occurred mostly in the upper extremity (47%) and trunk (34%). Lesions were sometimes multiple (40%) and recurrences (67%) were more common. Cases of PCMZL had an increase in reactive CD3+ T cells, with frequent PD1 expression, whereas cases of CD4+ TLPD often contained abundant reactive B cells. Twenty-five cases were identified as having overlapping features: 6 cases of PCMZL were clonal for both IG and TRG; 11 cases of CD4+ TLPD were clonal for IG and TRG and 6 cases of CD4+ TLPD had light chain restricted plasma cells. By NGS, 23 variants were detected in 15 genes, with PCMZL more likely to show alterations, most commonly affecting TNFAIP3 and FAS, altered in 5 cases. Both entities have an indolent clinical course with response to conservative therapy and management, and warrant interpretation as an LPD rather than overt lymphoma.

INTRODUCTION

Primary cutaneous lymphomas constitute a heterogonous group of non-Hodgkin lymphoma that occur in the skin with diverse clinical and biologic behavior. Primary cutaneous CD4+ small/medium T cell lymphoproliferative disorder (CD4+ TLPD) was included as a provisional entity in the 2018 World Health Organization–European Organization (WHO-EORTC) classification of cutaneous neoplasms (1). The lesions are usually solitary and typically occur in the head and neck region or upper trunk (2). Histologic examination often shows nodular to diffuse infiltrates involving the dermis (3). The infiltrates consist of small to medium pleomorphic T cells with less than 30% large cells. In addition to CD4, the abnormal lymphoid cells express follicular helper T cell (TFH) markers such as PD1, ICOS, BCL6 and CXCL13 (4, 5). CD4+ TLPD often have an indolent clinical course and an excellent prognosis. Only rare cases are associated with aggressive disease with extracutaneous dissemination (6, 7) Primary cutaneous marginal zone lymphoma was included in WHO classification of lymphomas under the category of extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) (8). However, the recently published International Consensus Classification (ICC) of Mature Lymphoid Neoplasms recommended segregation of this entity from other MALT lymphomas, based on distinctive phenotypic and clinical features, and rarity of involvement of sites outside the skin (9). The participants in the Clinical Advisory Committee led the ICC to propose a change in terminology to: Primary cutaneous marginal zone lymphoproliferative disorder (PCMZL). Multifocal lesions are more frequent in PCMZL and usually present in the upper extremities and sometimes the trunk (10). PCMZL exhibits a nodular to diffuse dermal infiltrate which is composed of small lymphocytes, marginal zone B-cells (centrocyte-like cells), plasmacytoid cells, and plasma cells, often associated with reactive follicles. Compared to CD4+ TLPD, cutaneous relapses are common, especially in patients presenting with multifocal skin lesions, but dissemination to extracutaneous sites is rarely observed (1). Two major variants have been recognized. One group expresses IgM, like most cases of MALT lymphoma, whereas cases with class-switched immunoglobulin are confined to the skin with usually only localized disease (11). About 13–40% of cases are IgG4 positive (12, 13).

There are cases of CD4+ TLPD and PCMZL that exhibit morphologic, immunophenotypic and molecular overlapping features. Few studies have reported PD1 expression in background T cells in PCMZL (14) and extracutaneous marginal zone lymphomas (15). This feature although not specific, is often seen in CD4+ TLPD and peripheral T-cell lymphomas (PTCLs) of follicular T cell derivation. Monotypic plasma cells are typically seen in PCMZL, but they have been rarely reported in CD4+ TLPD (16). In addition, dual lineage monoclonal B and T cell rearrangements have been described infrequently in both CD4+ TLPD (4) and PCMZL (17). Presence of these confounding features may lead to uncertainty in diagnosis. Here we conduct a comprehensive study to determine similarities and differences in the clinical and pathological features of PCMZL and CD4+ TLPD. We describe cases that show intermediate features between both entities. In addition, we apply genomic analysis using next generation sequencing (NGS) to determine if these lesions share genomic alterations or are distinct.

METHODS

Patient Selection

All cases were retrospectively retrieved from the pathology archives of the Laboratory of Pathology National Cancer Institute (NCI) from January 2005 to December 2020. Review was conducted under the auspices of a protocol approved by the Institutional Review Board of the NCI. From the database, we identified 56 cases of CD4+ TLPD and 38 cases of PCMZL. The diagnosis recorded for this study is the original diagnosis as rendered by the Hematopathology Section (ESJ/SP). Diagnostic criteria for CD4+ TLPD included patients with (1) single/multiple cutaneous lesions at diagnosis, with no extracutaneous involvement; (2) lymphocytic infiltrate composed of small and medium CD4+ T cells with <30% of large cells as defined by the WHO‐EORTC classification (1, 3) expression of at least 2 TFH markers among PD1, BCL6, ICOS and CD10. Patients with a diagnosis of mycosis fungoides or other T cell lymphomas with cutaneous involvement were excluded. Patients with PCMZL were included if the following criteria were met: (1) single/multiple cutaneous lesions at diagnosis, with no extracutaneous involvement; (2) lymphocytic infiltrate classified as PCMZL, established according to the criteria as defined by the WHO‐EORTC classification (1, 3). No documentation of a current or prior history of systemic marginal zone lymphoma or other B cell lymphoma. We assessed the following clinical data, including age at diagnosis, gender, site and number of lesions and recurrences.

Morphology and immunohistochemistry

Hematoxylin and eosin-stained sections were evaluated for the following: architectural pattern, location of the infiltrate and presence of epidermotropism. Immunohistochemical studies were performed on all cases with available material with CD3, CD4, CD8, CD20, PD1, MUM1, kappa and lambda. When possible, an expanded panel for additional TFH markers was performed with CD10, CXCL13, ICOS, PD1Stains for IgG, IgG4, IgM and IgA were performed on cases of PCMZL. Immunohistochemistry was performed on either a Ventana Benchmark automated immunostainer using Ultra View detection or Leica (BOND III) system. The panel of antibodies, clones, and sources are presented in Supplemental table 1. The expression of the different TFH markers was scored semi quantitatively and divided into 3 categories based on the percentage of positive cells in the whole T-cell infiltrate.

• -0: No staining

• -1: <5%

• -2: 5%−50%

• -3: >50%

At least 2 TFH markers (PD1, ICOS, BCL6, and CD10) were analyzed in all 56 samples, 3 TFH markers in 34 samples and 4 TFH in 11 samples. The proportion of T cells was estimated as the ratio of CD3+ cells to the total lymphocytic infiltrate. We also estimated the CD4/CD8 ratio to the whole T cell infiltrate and the proportion of B cells as the ratio of CD20+ cells to the total lymphocytic infiltrate, which included T-cells (CD3+), B-cells (CD20+), and plasma cells (IG+). Median percentages of expression were recorded. IgM, IgG, IgA and IgG4 was performed on cases of 36 cases of PCMZL. A positive stain for IgG4 was identified as presence of ≥40 IgG4 positive plasma cells per high-power field (HPF) or a ratio of IgG4 to IgG-positive plasma cells (IgG4: IgG) ≥ 40%.

Immunoglobulin Gene and T-Cell Receptor Gene Rearrangement Analysis

DNA was extracted from FFPE tissue sections using QIAGEN QIAamp DNA FFPE Tissue Kit on a QIAcube robotic system according to the manufacturer’s protocol (QIAGEN, Germantown, MD).Polymerase chain reaction (PCR) for immunoglobulin (IG) gene (IGH and IGK loci) and T-cell receptor (TRG locus) rearrangements was performed as previously described (15).

Next generation sequencing (NGS)

Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue sections from 26 patients, using the AllPrep DNA/RNA FFPE Kit or QIAmp DNA FFPE Tissue Kit/RNeasy FFPE kit (Qiagen). Libraries are prepared according to the manufacturer’s instructions provided with the TruSight ® Oncology 500 (TSO500, Illumina ®) kit, a capture-based next-generation sequencing (NGS) assay that analyzes 523 cancer relevant genes and assesses multiple variant types, including small nucleotide variants (SNVs), indels, and immunotherapy biomarkers that rely on analysis of multiple genomic loci, such as tumor mutational burden (TMB). Sequencing was performed using a NextSeq 550Dx system (Illumina ®). Proprietary TruSight Oncology 500 and TruSight Oncology 170 v1.0 local applications (Illumina ®) were used for alignment, variant calling, gene fusion detection and for determination of TMB and MSI. All alignments were performed utilizing Illumina designed browser extensible data (BED) files that are based on the human genome reference GRCh37/hg19 assembly. The resultant variant call files (VCF) files were uploaded to our in-house webtool, the Oncogenomics database (NIH, Bethesda MD) for filtering and analysis. Filtering parameters included removal of single nucleotide polymorphisms (SNPs) from population databases (1000 genomes project, gnomAD, ExAC, etc) with a maximum population allele frequency of ≥0.002, ≥5 supporting reads in the tumor, and a variant allele frequency (VAF) ≥ 0.02. Intronic, UTRs, intergenic locations and repetitive regions were excluded. The variants were manually reviewed to exclude sequencing artifacts using the Integrative Genomics Viewer (Broad Institute, Cambridge, MA). From this point, each variant was considered based on supporting data (COSMIC, cBioPortal, in-house database, VAF compared to expected tumor content) to be included as pathogenic or likely pathogenic variants.

Statistical Analysis

Statistical analysis was performed using GraghPad Prism software (version 9; San Diego, CA). Continuous data were recorded as median (interquartile range, IQR). Categorical parameters were reported as frequencies and associated percentages. Comparisons between groups for continuous data were performed using the Student t test. For categorical data, Fisher exact tests were performed. The statistical significance of the analyses was confirmed when a P value was less than 0.05.

RESULTS

Clinical characteristics of the study population

From an initial selection of 90 patients with CD4+ TLPD and 76 patients with PCMZL from our database, 34 patients and 38 patients were excluded respectively due to lack of material for additional immunostaining and/or molecular studies. Finally, 56 CD4+ TLPD patients were included (36 males, 20 females). Median age was 53 years [IQR; 37.5–64.0], ranging from 4 to 85 years (Table 1). Thirty-eight patients were diagnosed as PCMZL (20 females, 18 males) with a median age of 58 [IQR; 38–73] years and a range of 11–89 years. The lesions of CD4+ TLPD were mainly located on the head and neck (n=36) followed by the trunk (n=12) and upper extremities. The most frequent sites involved by PCMZL were the upper extremities (47%), trunk (34%) and leg (18%). A total of 51 CD4+ TLPD patients (91%) had solitary lesions and two of the patients had recurrences. In contrast 40% patients with PCMZL had bifocal or multiple lesions and recurrences were reported in 16 patients.

Table 1.

Clinical characteristics of patients with PCMZL and CD4+ TLPD.

	PCMZL	CD4+ TLPD	P value
NUMBER OF PATIENTS	38	56
AGE	58 (38–73)	53 (37.5–64)	0.34
SEX
Male	18	36	0.14
Female	20	20
SITE OF LESION
Upper extremity	18 (47%)	9 (17%)	0.0021
Trunk	13 (34%)	12 (21%)	0.23
Head and neck	5 (13%)	36 (64%)	<0.0001
Abdomen	2 (5%)	3 (5%)	>0.9999
Leg	7 (18%)	0 (0%)	0.0012
NUMBER OF SITES
Solitary	23 (60%)	51 (91%)	0.0006
Bifocal	9 (24%)	4 (7%)	0.03
Multiple	6 (16%)	1 (2%)	0.016
RECURRENCES	16/24 (67%)	2/27 (7%)	<0.0001

PCMZL; Primary cutaneous marginal zone lymphoma, CD4+ TLPD; Primary cutaneous CD4 positive small/medium T cell lymphoproliferative disorder

significant P value< 0.05

Histopathologic features

Four architectural patterns were observed in CD4+ TLPD, namely nodular, diffuse, nodular and diffuse and band-like, whereas nodular, diffuse as well as nodular and diffuse infiltrates were detected in PCMZL (Table 2). Twenty-five CD4+ TLPD cases (45%) were characterized by a dense, nodular lymphoid infiltrate (Figure 1A), 7 cases (12%) revealed a diffuse infiltrate (Figure 1B), 23 patients had a nodular and diffuse pattern (Figure 1C), while 1 patient had a band-like infiltrate (Figure 1D). The cases of CD4+ TLPD typically demonstrated small to medium atypical lymphoid cells (Figure 1E) with few scattered large lymphoid cells, admixed with reactive lymphocytes, histiocytes and plasma cells. In cases with PCMZL, the lesions presented mostly as either a nodular infiltrate (60%) (Figure 1F) or a nodular and diffuse pattern (37%) (Figure 1G). The diffuse pattern (Figure 1H) was less frequent (3%) and band like infiltrates were absent. At higher power, PCMZL (Figure 1I) contained a moderately dispersed infiltrate of small to medium sized lymphocytes, plasmacytoid cells and plasma cells. The lymphoid infiltrates of CD4+ TLPD and PCMZL involved the dermis extending to the subcutis in 52% and 58% of cases respectively (p = 0.51). Significant epidermotropism was not seen in either CD4+ TLPD or PCMZL.

Table 2.

Histologic and phenotypic characteristics of patients with PCMZL and CD4+ TLPD.

	PCMZL	CD4+ TLPD	P value
NUMBER OF PATIENTS	38	56
ARCHITECTURAL PATTERN
Nodular	23 (60%)	25 (45%)	0.15
Diffuse	1 (3%)	7 (12%)	0.14
Nodular and diffuse	14 (37%)	23(41%)	0.83
Band-like	0 (0%)	1 (2%)	>0.999
INFILTRATE LOCATION
Subepidermal/Superficial	3 (8%)	4 (7%)	>0.999
Superficial and deep dermis	13 (34%)	23 (41%)	0.50
Dermis and subcutis	22 (58%)	29 (52%)	0.51
EPIDERMOTRPISM
Mild	2 (5%)	5 (89%)	0.70
Pautrier abscesses (<3)	0 (0%)	2 (5%)	0.51
Prominent	0 (0%)	0 (0%)	>0.999
IMMUNOHISTOCHEMISTRY
PD1
Median	10 (5–30)	40 (20–50)	<0.001
< 5%	4 (11%)	0 (0%)	0.02
5–50%	33 (86%)	49 (87%)	0.9
>50%	1 (3%)	7 (13%)	0.04
CD3+ T cells (median)	45 (40–67.5)	70 (60–80)	0.001
CD4+ T cells (median)	70 (60–80)	80 (70–90)	0.05
CD8+ T cells (median)	30 (20–40)	20 (10–30)	0.05
CD20+ B cells (median)	55 (32.5–60)	30 (20–40)	0.001
MUM1 + plasmacytoid cells (median)	10 (5–20)	10 (5–10)	0.32
kappa	17/38 (45%)	3/56 (5%)	<0.0001
lambda	16/38 (42%)	6/56 (11%)	0.0009
Polytypic plasma cells	5/38 (13%)	47/56 (84%)	<0.0001

PCMZL; Primary cutaneous marginal zone lymphoma, CD4+ TLPD; Primary cutaneous CD4 positive small/medium T cell lymphoproliferative disorder

significant P value< 0.05

Figure 1.

Morphologic features of CD4+TLPD and PCMZL. Morphologic presentation of CD4+TLPD can occur as (A) nodular (B) diffuse (C) nodular and diffuse or (D) band-like infiltrates that are composed of (E) small/medium atypical lymphoid cells admixed with histiocytes, reactive lymphocytes and plasma cells. Cases of PCMZL present as (F) nodular, (G) nodular and diffuse and (H) diffuse infiltrates with (I) atypical small to medium lymphoid cells. PCMZL; Primary cutaneous marginal zone lymphoma, CD4+TLPD; primary cutaneous CD4+ small/medium T cell lymphoproliferative disorder.

Immunophenotypic features

CD4+ TLPD was characterized by the presence of a T-cell component (median of CD3 expression=70% [IQR, 60 to 90], range, 50–90). PD1, ICOS, and BCL6 expression was analyzed (Supplemental table 2). PD1 expression was present in all samples, whereas ICOS (median=5% [IQR, 3 to 10]), BCL6 (median=5% [IQR, 1 to 10]) and CD10 (median=2% [IQR, 1 to 5]) were less frequently expressed in a subset of the T cells. Although cases of CD4+ TLPD showed significantly higher proportions of CD3+ T (p=0.001) and PD1+ (P < 0.001) T cells when compared to PCMZL, in neither disease entities did we observe any difference in proportion of PD1+ T cells with an expression between (<5–50%) (p=0.9). PCMZL cases had higher frequencies of B cells (P=0.001), but MUM1 expression rate showed no differences between the CD4+ TLPD and PCMZL (P=0.32). However, monotypic plasma cells occurred more frequently (89%) in PCMZL. In addition, the majority of the plasma cells in PCMZL were positive for IgG (94%) and 12% were positive for IgG4 (Supplemental table 3). Monotypic plasma cells were seen in 9 patients (16%) with CD4+TLPD.

Clonality Studies

Clonal IG gene rearrangements were present in 28 cases (76%) of PCMZL, while 1 case was suspicious for both IG and TRG gene rearrangement (Table 3). Eight cases of PCMZL were clonal for TRG, whereas three cases had peaks that were suspicious for TRG. Clonal IG gene rearrangements were detected in 6 cases with TRG gene rearrangements. Six cases had polyclonal IG and TRG gene rearrangements. On the other hand, the majority of the CD4+ TLPD cases (89%) were positive for clonal TRG gene rearrangement, 1 case had an oligoclonal TRG, 1 case was suspicious for TRG and only 4 cases had polyclonal TRG gene rearrangements. Four cases had peaks of increased intensity that were suspicious for IG gene rearrangement, whereas 11 cases of CD4+ TLPD had clonal IG. The cases with clonal IG also had clonal TRG gene rearrangements.

Table 3.

Molecular clonality of PCMZL and CD4+ TLPD cases by PCR

	PCMZL	CD4+ TLPD	P value
NUMBER OF PATIENTS	37	56
PCR CLONALITY
Ig clone only	19 (51%)	0 (0%)	<0.0001
TCRG clone only	2 (6%)	37 (66%)	<0.0001
Clonal Ig and TCRG clones	6 (16%)	10 (18%)	0.55
Ig and Oligoclonal TCRG	0 (0%)	1 (2%)	>0.999
Clonal Ig and suspicious TCRG	3 (8%)	0 (0%)	0.51
Clonal TCRG and suspicious Ig	0 (0%)	3 (5%)	0.51
Suspicious Ig and PC TCRG	1 (3%)	1 (2%)	>0.999
Suspicious TCRG and PC Ig	0 (0%)	1 (2%)	>0.999
Polyclonal for Ig and TCRG	6 (16%)	3 (5%)	0.14

Ig; Immunoglobulin, TCRG; T cell receptor gamma, PC; Polyclonal, PCMZL; Primary cutaneous marginal zone lymphoma, CD4+ TLPD; Primary cutaneous CD4 positive small/medium T cell lymphoproliferative disorder.

significant P value< 0.05

Cases with overlapping features

Because monotypic plasma cells and clonal dual lineage gene rearrangement were identified in both PCMZL and CD4+ TLPD, we sought to identify cases with overlapping features that may present a diagnostic challenge. Twenty-five cases (Table 4) were identified as having overlapping features between PCMZL and CD4+ TLPD and included 7 females and 18 males with a median age of 64 [IQR, 46–74.5] years, ranging from 4–86 years. Twenty patients (80%) had solitary lesions, localized to the head region in 12 (48%) and to other body areas such as the trunk (20%) and upper extremities (UE) (16%) in 13 patients. Eight cases were diagnosed as PCMZL, whereas 17 cases had CD4+ TLPD. Eighty-eight percent of the cases had a PD1 expression score of 2, while the rest of the cases had mostly a score of 3. Six cases of PCMZL showed both IG and TRG rearrangement. The cases were characterized by atypical CD20+ B cells and were rich in reactive CD3+ and CD4+ T cells (Figure 2). PD1 was positive in a subset of the T cells. The plasma cells were light chain restricted. Both IG and TRG peaks were identified by PCR. Other cases with intermediate features included 2 cases of PCMZL with TRG gene rearrangement and lambda light chain restricted plasma cells; 10 cases of CD4+ TLPD with both clonal B cell and clonal T cell rearrangement, one case with clonal IG and oligoclonal T cell gene rearrangement and 6 cases of CD4+ TLPD with T clones but had either kappa or lambda light chain restricted plasma cells. Three cases of CD4+ TLPD that had both B and T clones also had monotypic plasma cells (Figure 3).

Table 4.

Summary of features in cases with overlapping features of PCMZL and CD4+ TLPD

Case #	Age (yr)	Sex	Site	No Lesions	PD1 (%)	Light chain	IG	TRG	Diagnosis
1	78	M	UE	1	40	lambda	IGH, IGK	clonal	PCMZL
2	69	F	Head	2	40	lambda	IGH, IGK	clonal	PCMZL
3	77	F	Trunk	2	10	kappa	IGH, IGK	clonal	PCMZL
4	64	F	UE/buttock	2	10	kappa	IGH	clonal	PCMZL
5	86	M	Trunk	1	40	kappa	IGH, IGK	clonal	PCMZL
6	66	M	Trunk	1	30	kappa	IGH	clonal	PCMZL
7	69	F	Trunk/UE	3	50	lambda	polyclonal	clonal	PCMZL
8	55	F	Leg	1	1	lambda	polyclonal	clonal	PCMZL
9	79	M	Head	1	30	polytypic	IGH, IGK	clonal	CD4+ TLPD
10	79	M	UE	1	20	polytypic	IGH	clonal	CD4+ TLPD
11	71	M	UE/trunk	2	60	polytypic	IGH	clonal	CD4+ TLPD
12	85	M	Head	1	50	lambda	IGH, IGK	clonal	CD4+ TLPD
13	4	F	trunk	1	50	kappa	IGH, IGK	clonal	CD4+ TLPD
14	52	M	Head	1	30	polytypic	IGH, IGK	clonal	CD4+ TLPD
15	40	M	Head	2	40	polytypic	IGH, IGK	clonal	CD4+ TLPD
16	34	M	trunk	1	50	kappa	IGH	clonal	CD4+ TLPD
17	64	M	Head	1	30	polytypic	IGH, IGK	clonal	CD4+ TLPD
18	33	M	Head	1	50	Polytypic	IGH	clonal	CD4+ TLPD
19	55	M	Head	1	10	polytypic	IGH	oligoclonal	CD4+ TLPD
20	32	M	Head	1	70	lambda	indeterminate	clonal	CD4+ TLPD
21	61	M	Head	1	80	lambda	polyclonal	clonal	CD4+ TLPD
22	63	M	UE	1	40	lambda	polyclonal	clonal	CD4+ TLPD
23	62	M	Head	1	40	lambda	polyclonal	clonal	CD4+ TLPD
24	19	F	UE	1	40	lambda	polyclonal	clonal	CD4+ TLPD
25	72	M	Head	1	20	kappa	polyclonal	clonal	CD4+ TLPD

Age, Age at diagnosis; Yr, year; M, male; F, female; #, number; UE, upper extremity; TRCG, T cell receptor gamma; IG, immunoglobulin; PCMZL, primary cutaneous marginal zone lymphoma; CD4+ TLPD, primary cutaneous CD4 positive small/medium T cell lymphoproliferative disorder.

Figure 2.

PCMZL with overlapping features, Case 2. Skin biopsy (A), hematoxylin and eosin) shows a dense dermal lymphoid infiltrate. Numerous lymphoid cells are positive for CD 20, with numerous admixed T-cells with CD3. CD4 stains the majority of the cells, with many cells positive for PD1. Numerous plasmacytoid cells are positive for MUM1, and double-staining shows largely restricted staining for lambda light chain (red), over kappa (brown). Clonal peaks are seen with IG and IGK PCR. However, increased peaks in a polyclonal background are seen with TRG.

Figure 3.

CD4+TLPD with overlapping features, Case 12. A dense dermal lymphoid infiltrate is positive for CD3 and CD4 with numerous cells positive for PD1. CD20 stains nodular and diffuse B cells. MUM1 stains plasmacytoid cells that are negative for kappa but positive for lambda. PCR studies show a clonal IGH gene rearrangement with FRI and FRIII. TRG shows several increased peaks.

Genomic Profiling

NGS was performed in 19 patients with CD4+ TLPD and 7 patients with PCMZL to investigate overlapping and differing mutational profiles as well as to further examine the molecular pathogenesis of these entities. 4 patient samples (3 CD4+ TLPD and 1 PCMZL) failed to produce sufficient DNA for amplification leaving 16 cases of CD4+ TLPD and 6 cases of PCMZL for analysis. The median exon coverage ranged from 391–1374 and percent of exons covered by at least 50 reads ranged from 98.8–99.5%. A total of 23 variants classified as pathogenic/likely pathogenic were detected in 15 genes (Figure 4, Supplemental Table 4). The average variant allele fraction (VAF) of these variants was 0.047 for CD4+ TLPD and 0.085 for PCMZL. The most frequently detected variants in CD4+ TLPD were in epigenetic regulation while PCMZL showed mutations most frequently in NF-κB, specifically in TNFAIP3. EP300 was the only gene to show alterations in both entities with one variant detected in CD4+ TLPD and in PCMZL. Variants of TNFAIP3 and FAS were detected in 50% (3/6) and 33% (2/6) cases of PCMZL respectively. No genes showed alterations in multiple cases of CD4+ TLPD.

Figure 4.

Oncoplot showing the distribution of variants across case samples of CD4+ TLPD and PCMZL. Associated pathways are shown to the left of affected genes. The bar graph across the top shows the number of variants per sample and the bar graph along the right shows the number of variants per gene. The colors indicate the type of genetic alteration as shown below the oncoplot. Overall, there is limited overlap between the two types of lesions. Variants were more frequently detected in PCMZL samples, which may partially be due to tumor content and panel design.

Outcome

Follow up and outcome information was available for 27 patients with CD4+ TLPD and 24 patients with PCMZL. Median follow-up for the CD4+ TLPD cases was 20 months [9–26 months] (range, 3 to 84 months). Twenty five of the 27 patients for whom follow-up data were available achieved a complete remission. Two patients had a recurrent lesion, one at 3 months and the other at 9 months. One patient presented with a solitary lesion and the other had 3 lesions. Of the two cases that recurred, one had both B and T clones and the other had a B cell and oligoclonal T cell gene rearrangement. Management of the lesion included surgical excision for all patients. In addition, one patient received intralesional steroids. Median follow-up for the PCMZL patients was 24 months, [12–78] (range, 1 to 132). Of the 24 patients for whom follow-up data was available, seven achieved complete remission after treatment without relapse. Sixteen patients had a recurrent lesion, with median time to first relapse of 30 months [13–60 months] (range, 4 to 96 months). Twelve patients had only one relapse, 3 patients had 2 recurrent lesions, whereas one patient had 3 relapses. Of the lesions that recurred, eight were solitary lesions, 3 bifocal lesions and 5 multiple lesions. Two of these cases had both B and T clones and two had a polyclonal B cell and a clonal T cell gene rearrangement. One patient had clonal Ig and abnormal TRG gene rearrangement, whereas, two patients had polyclonal Ig and TRG gene rearrangements. The remaining patients had a clonal B and polyclonal T cell gene rearrangement. Management of the lesion ranged after biopsy from surgical excision (n=14 [50%]), excision and local radiation (n=4 [18.2%]), chemotherapy with rituximab (n=2 [9.1%]), excision and rituximab (n=1[4.5%]), chemotherapy alone (n=1), chemotherapy and local radiation (n = 1), topical steroids (n = 1).

DISCUSSION

Traditional diagnostic criteria propose that CD4+ TLPD and PCMZL can be differentiated based on the location, number of lesions and the immunophenotype of the tumor cells. In CD4+ TLPD, the lesions are usually solitary and occur primarily in the head and neck region with the neoplastic T cells positive for TFH markers. In contrast, multiple lesions are more frequent in PCMZL, usually presenting in the upper extremities and characterized by monotypic plasma cells. Both diseases are usually indolent and can be treated conservatively. However, PCMZL can present with multiple/recurrent lesions which may require additional treatment with rituximab or chemotherapy.

In this study we detected a significant number of cases with diagnostic overlap between the two entities. Cases of CD4+ TLPD are frequently rich in B-cells. More unexpected is the presence of light chain restricted plasma cells or clonal B and T cell gene rearrangements. Although PD1 expression, is typically observed in CD4+ TLPD, increased expression of this marker also was detected in PCMZL. In prior work we reported expanded numbers of TFH cells in nodal marginal zone lymphoma (15). Monoclonal and oligoclonal T cell populations may be detected in a variety of nonneoplastic conditions, including immunodeficiency syndromes, autoimmune disease, post T-cell ablative therapies, post bone marrow transplantation, and as unusual responses to viral infections (18–20). Clonal T-cell proliferations can occur as part of a normal immune response, are thought to be antigen driven (21) and may explain the presence of T cell clones without morphologic evidence of a T cell malignancy in a B cell lymphoma. In addition, NGS approaches to identify clonality offer greater specificity than PCR-based methods for distinguishing a true clonal expansion from increased peaks of uncertain significance(22). This methodology is not yet in routine use in most hematopathology laboratories.

Some authors have suggested that primary cutaneous CD4+ TLPD should be considered a form of “pseudolymphoma” and that it might represent an exaggerated local immune response to an unknown antigenic stimulus. Based on its benign clinical course and indolent behavior, this entity was “downgraded” to the status of a lymphoproliferative disorder (LPD) rather than lymphoma in the 2017 WHO classification (23). Its definition and status remain unchanged from 2017 in the ICC proposal. Similarly, primary cutaneous marginal zone lymphoma shows major differences with MALT lymphomas in other anatomic sites and led the ICC to propose a similar change in terminology to Primary cutaneous marginal zone lymphoproliferative disorder (PCMZL), a term we endorse using in routine practice (9, 24). The lesions nearly always remain confined to the skin. The presence of clonal plasma cells with evidence of class switch also supports that like CD4+ TLPD, PCMZL may be better categorized as a lymphoproliferative disorder rather than lymphoma. In most instances the precipitating antigen is unknown, but some cases have been linked to infection with Borrelia burgdorferi (25, 26). Our study indicating that CD4+ TLPD and PCMZL frequently show overlapping features supports the view that an exaggerated immune response may be one of the causative factors in both conditions, although many other aspects indicate that they are distinct entities. A low level of genomic complexity is seen in both conditions, which is also supportive of the designation as “lymphoproliferative disorders”.

We used NGS to successfully analyze 16 cases of CD4+ TLPD and 6 cases of PCMZL which yielded 23 variants. Variants were more frequently detected in PCMZL (14 variants for 6 cases) than CD4+ TLPD (9 variants for 16 cases). The average VAF for CD4+ TLPD was 0.047 compared to 0.085 for PCMZL which suggests a lower tumor content on average and potentially contributed to fewer detected variants detected for CD4+ TLPD. The molecular profiling shows EP300 as the lone overlapping variant between PCMZL and CD4+ TLPD. This indicates separate pathologic drivers, as was expected, despite the overlapping morphology in these cases. CD4+ TLPD showed variants involved with epigenetic regulation (ARID1A, EP300, SETD2) but did not otherwise show a discernable pattern. For the PCMZL cases, recurrent variants were detected in FAS (2/6, 33% of cases) and in TNFAIP3 (3/6, 50%). Alterations of TNFAIP3 have been previously identified in ocular marginal zone lymphoma with a reported frequency as high as 54%.(27, 28) Gene set enrichment analysis demonstrated upregulation of NF-κB in these ocular marginal zone lymphomas compared to marginal zone B-cells, indicating the significance of this pathway. TNFAIP3 is a key negative regulator of the NF-κB pathway and has been identified as a tumor suppressor gene in classic Hodgkin lymphoma and primary mediastinal B-cell lymphoma (29). In previous series, FAS alterations were the most frequently detected variant in PCMZL (30). We detected a frameshift mutation slightly upstream of the death domain and a point mutation occurring within the death domain, both of which have been shown to have an anti-apoptotic effect (31, 32).

Unlike CD4+ TLPD, the cytogenetic and other molecular features of PCMZL have been evaluated in some previously published work. Cytogenetic and FISH abnormalities, including t(3;14)(p14.1;q32) involving the IGH and FOXP1 genes have been detected in a significant proportion of PCMZL (33) and are separate from those seen in other MALT lymphoma, which are partly defined by t(11;18)(q21;q21) and t(1;14)(p22;q32) (34, 35). Our results also show some differences with mutations reported in extranodal MALT lymphoma(36). Recurrent mutations in CD4+ TLPD have been poorly defined. Beltzung and coworkers (4) detected a mutation in DMNT3A gene in a case of CD4+ TLPD, but recurrent mutations in DMNT3A or any other gene have not been identified in the literature. In our cohort, we identified mutations in ARID1A, ATM and TP53 genes in one case of CD4+ TLPD. Although mutations in these genes have been described in T cell lymphomas such as mycosis fungoides, they have also been demonstrated in B cell lymphomas. SETD2, a chromatin modifying gene, is commonly mutated in T cell lymphomas, including hepatosplenic T cell lymphoma (37) and monomorphic epitheliotropic intestinal T cell lymphoma (38). We observed a deletion in SETD2 gene in a single case of CD4+ TLPD with both B and T clones, which though non-recurrent, was helpful in suggesting a T cell origin. Mutations in NF1 can occur in non-Hodgkin lymphoma and its detection in one case of CD4+ TLPD is of uncertain significance. Given that these were isolated events in single cases, suggests caution in interpreting their significance.

Because cases with overlapping features of CD4+ TLPD and PCMZL can present a diagnostic challenge, we suggest criteria that can aid in this distinction in routine practice (Table 5). Cases presenting in the upper extremity with multiple or recurrent lesions favor a diagnosis of PCMZL. In addition, identifying IgG/IgG4 positive light chain restricted plasma cells with a B cell clone further supports the diagnosis (12, 13). In contrast, a solitary lesion occurring in the head and neck region with T cells expressing TFH markers with a T cell clone favors a diagnosis of CD4+ TLPD. Indeed, using these factors, case 2, originally classified as PCMZL, is now favored as CD4+ TLPD based on the clinical presentation as a solitary lesion involving the head. If a diagnosis is still indeterminate, NGS ultimately may provide further information.

Table 5.

Suggested Criteria for the Differential Diagnosis of PCMZL and CD4+ TLPD

FAVORS DIAGNOSIS OF PCMZL

Location in upper extremities

Multiple lesions

Recurrent lesion

Reactive follicles

Light chain restricted plasma cells

IgG/IgG4 positive plasma cells

Clonal Immunoglobulin gene rearrangement and negative TRG gene rearrangement

Mutations in FAS and NFkB pathway genes

FAVORS DIAGNOSIS OF CD4+ TLPD

Location in head and neck

Solitary lesion

No recurrence

Nodular aggregates of B-cells

Mutations in DMNT3A (rare)

FEATURES COMMON TO BOTH ENTITIES

Increased TFH cells

Solitary lesion

Ambiguous clonality studies

Rare pathogenetic mutations

Benign clinical course

PCMZL, primary cutaneous marginal zone lymphoma; CD4+ TLPD, primary cutaneous CD4 positive small/medium T cell lymphoproliferative disorder; TRG, T cell receptor gamma.

In conclusion, although cases of CD4+ TLPD and PCMZL show overlapping features which can lead to problems in differential diagnosis, the final interpretation should be made based on clinical and pathologic features. Future studies with deep sequencing may reveal recurrent genetic alterations that may be helpful in the differentiation of these two clinical entities.

Supplementary Material

Supplemental Data File (.doc, .tif, pdf, etc.)_1

Supplemental Table 1. Antibodies used in the immunophenotypic analysis

Supplemental Data File (.doc, .tif, pdf, etc.)_2

Supplemental Table 2. Follicular helper T cell expression in CD4+ TLPD cases.

Supplemental Data File (.doc, .tif, pdf, etc.)_3

Supplemental Table 3. Heavy chain expression in PCMZL cases.

Supplemental Data File (.doc, .tif, pdf, etc.)_4

Supplemental Table 4. Molecular alterations identified in CD4+ TLPD and PCMZL