Fourteen PCBCL, including PCFCL and PCDLBCL-LT, collected over a 7-year period (January 2011 to December 2017) at the Pathology Department of the University of Salerno (Italy), were retrieved and evaluated together with 14 nDLBCL used as controls. PCMZL cases were not included in the present study.
Cutaneous and lymph nodal biopsies were available for all the samples. All the corresponding patients had received a diagnosis of PCBCL on longstanding single or multiple cutaneous lesions from different sites (Tab. 1).
Table 1
Clinical, histologic, phenotypic and gene expression profiling of 14 PCBCL.
Patients
|
1c
|
2c
|
3c
|
4c
|
5c
|
6c
|
7c
|
Sex
|
M
|
M
|
M
|
F
|
F
|
M
|
M
|
Age
|
65
|
54
|
76
|
54
|
56
|
80
|
49
|
Site
|
Trunk
|
Eyelid
|
Arm
|
Leg
|
Leg
|
Arm
|
Trunk
|
Size, mm
|
44
|
18
|
23
|
15
|
25
|
10
|
12
|
Relapse
|
Yes (LN cervical)
|
Yes (Local)
|
Yes (Local)
|
Yes (Local)
|
No
|
No
|
No
|
Diagnosis
|
PCDLBCL-LT
|
PCFCL
|
PCFCL
|
PCDLBCL-LT
|
PCDLBCL-LT
|
PCFCL
|
PCFCL
|
Reactive T-cells
|
Yes
|
Yes
|
Yes
|
Yes
|
No
|
No
|
Yes
|
Growth pattern
|
Diffuse
|
Follicular
|
Diffuse
|
Diffuse
|
Diffuse
|
Follicular
|
Follicular
|
Dendritic meshwork
|
No
|
Yes
|
Yes
|
No
|
No
|
No
|
No
|
Skin ulceration
|
No
|
No
|
No
|
No
|
Yes
|
No
|
No
|
Necrosis
|
No
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
No
|
Nuclear debris
|
No
|
No
|
Yes
|
Yes
|
Yes
|
Yes
|
No
|
“Starry sky” pattern
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Ki67 positivity (%)
|
80%
|
20%
|
80%
|
60%
|
90%
|
60%
|
60%
|
CD2
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD4
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD5
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD8
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD3
|
+
|
+
|
+
|
+
|
-
|
-
|
+
|
CD20
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
CD79A
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
PAX5
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Alk
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CyclinD1
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Cytokeratin
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
MYC
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD30
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD68
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD138
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Bcl2
|
+
|
+
|
-
|
+
|
+
|
+
|
+
|
Bcl6
|
+
|
+
|
+
|
-
|
-
|
+
|
+
|
CD10
|
-
|
+
|
+
|
-
|
-
|
+
|
+
|
Mum1/IRF4
|
+
|
-
|
-
|
+
|
+
|
-
|
-
|
Hans’ algorithm
|
Non-GCB
|
GCB
|
GCB
|
Non-GCB
|
Non-GCB
|
GCB
|
GCB
|
Colomo’s algorithm
|
Non-GCB
|
GCB
|
GCB
|
Non-GCB
|
Non-GCB
|
GCB
|
GCB
|
Muris’ algorithm
|
Non-GCB
|
GCB
|
GCB
|
Non-GCB
|
Non-GCB
|
GCB
|
GCB
|
t(14;18)-PCR
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
GEP
|
ABC
|
GCB
|
GCB
|
Unclassified
|
ABC
|
GCB
|
GCB
|
|
|
|
|
|
|
|
|
Patients
|
8c
|
9c
|
10c
|
11c
|
12c
|
13c
|
14c
|
Sex
|
F
|
M
|
F
|
M
|
F
|
F
|
F
|
Age
|
80
|
76
|
45
|
58
|
73
|
40
|
67
|
Site
|
Arm
|
Leg
|
Head
|
Trunk
|
Trunk
|
Trunk
|
Arm
|
Size, mm
|
30
|
23
|
14
|
28
|
16
|
9
|
21
|
Relapse
|
Yes (local)
|
No
|
No
|
Yes (local)
|
No
|
No
|
No
|
Diagnosis
|
PCFCL
|
PCDLBCL-LT
|
PCFCL
|
PCFCL
|
PCFCL
|
PCFCL
|
PCFCL
|
Reactive T-cells
|
No
|
No
|
Yes
|
No
|
No
|
Yes
|
No
|
Growth pattern
|
Diffuse
|
Diffuse
|
Mixed
|
Diffuse
|
Mixed
|
Follicular
|
Mixed
|
Dendritic meshwork
|
No
|
No
|
Yes
|
Yes
|
No
|
No
|
No
|
Skin ulceration
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Necrosis
|
Yes
|
No
|
No
|
Yes
|
No
|
No
|
No
|
Nuclear debris
|
Yes
|
No
|
No
|
Yes
|
No
|
No
|
No
|
“Starry sky” pattern
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Ki67 positivity (%)
|
87%
|
75%
|
30%
|
78%
|
82%
|
65%
|
80%
|
CD2
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD4
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD5
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD8
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD3
|
-
|
-
|
+
|
-
|
-
|
+
|
-
|
CD20
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
CD79A
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
PAX5
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Alk
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CyclinD1
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Cytokeratin
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
MYC
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD30
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD68
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CD138
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Bcl2
|
+
|
+
|
-
|
+
|
+
|
-
|
-
|
Bcl6
|
-
|
-
|
+
|
+
|
+
|
+
|
+
|
CD10
|
-
|
-
|
+
|
-
|
-
|
+
|
+
|
Mum1/IRF4
|
+
|
+
|
-
|
+
|
+
|
-
|
-
|
Hans’ algorithm
|
Non-GCB
|
Non-GCB
|
GCB
|
Non-GCB
|
Non-GCB
|
GCB
|
GCB
|
Colomo’s algorithm
|
Non-GCB
|
Non-GCB
|
GCB
|
Non-GCB
|
Non-GCB
|
GCB
|
GCB
|
Muris’ algorithm
|
Non-GCB
|
Non-GCB
|
GCB
|
Non-GCB
|
Non-GCB
|
GCB
|
GCB
|
t(14;18)-PCR
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
GEP
|
ABC
|
ABC
|
GCB
|
ABC
|
ABC
|
GCB
|
GCB
|
LN: lymph node; PCBCL: primary cutaneous large, B-cell lymphoma; PCFCL: primary cutaneous follicle centre lymphoma; PCDLBCL-LT: primary cutaneous diffuse, large, B-cell lymphoma, leg type; ABC: Activated-B-Cell; GCB: Germinal Center-B-Cell; IHC: immunohistochemistry; GEP: Gene Expression Profiling. |
The pathological history in all the patients was negative for previous lymphoma.
After the histological diagnosis clinical staging was assessed in each of them by flow cytometry of peripheral blood, immunoglobulin gene rearrangements in lesional skin or in peripheral blood, complete and differential blood cell count, routine serum biochemistry analysis with lactate dehydrogenase (LDH), serum protein electrophoresis, liver function tests, computed tomography-fluorodeoxyglucose-positron emission tomography (CT-FDG-PET) scan and bone marrow biopsy. The staging and therapy were assessed according to the European Society for Medical Oncology (ESMO) recommendations for primary cutaneous lymphoma [13]. All the patients, after specific therapies, underwent strict clinical follow-up.
Immunohistochemistry
Immunohistochemistry (IHC) was performed on 4-µm serial sections of formalin-fixed, paraffin-embedded (FFPE) tissue from each sample using CD20, CD79A, PAX5, CD10, BCL6, MUM1/IRF4, BCL2, CD3, CD68, CD5, CD2, CD4, CD8, CD138, CD30, ALK, CyclinD1, MYC, Cytokeratin and Ki-67.
The sections were stained using the BenchMark XT autostainer (Ventana Medical Systems Inc, Tucson, Arizona) with standard protocols [14-16]. The following pre-diluted monoclonal antibodies were used in all cases: CD20 (1:100; clone L26; cat. N. 760-2531; Ventana); CD79a (1:100; clone SP18; cat. N. 790-4432; Ventana); PAX5 (1:100; clone SP34; cat. N. 790-4420; Ventana); CD10 (1:25; clone SP67; cat. N. 790-4506, Ventana), BCL6 (1:200; clone GI191/A8; cat. N. 760-4241, Cell Marque Corporation – Sigma Aldrich, Rocklin, California); Mum1/IRF4 (1:100; clone MRQ-43; cat. N. 760-4529; Cell Marque Corporation); BCL2 (1:100; clone 124; cat. N. 790-4464; Ventana); CD3 (1:100; clone 2GV6; cat. N. 790-4341; Ventana); CD68 (1:100; clone KP-1; cat. N. 790-2931; Ventana); CD5 (1:100; clone SP19; cat. N. 790-4451; Ventana); CD2 (1:100; clone MRQ-11; cat. N. 760-4377; Ventana); CD4 (1:100; clone SP35; cat. N. 790-4423; Ventana); CD8 (1:100; clone SP57; cat. N. 790-4460; Ventana); CD138 (1:100; clone B-A38; cat. N. 760-4248; Ventana); CD30 (1:100; clone Ber-H2; cat. N. 790-2926; Ventana); ALK (1:100; clone D5F3; cat. N. 790-4794; Ventana); CyclinD1 (1:100; clone SP4-R; cat. N. 790-4508; Ventana); MYC (1:100; clone Y69; cat. N. 790-4628; Ventana); Cytokeratin (1:100; clone AE1/AE3/PCK26; cat. N. 760-2595; Ventana) and Ki-67 (1:100; clone 30-9; cat. N. 790-4286; Ventana). The slides were then washed and incubated with multimer-labeled anti-mouse or rabbit IgG (cat. N. 760-2680, Ventana). Staining was then performed using the iVIEW 3,3’-diaminobenzidine detection kit (Ventana) according to the manufacturer’s instructions. Samples were then counterstained with Hematoxylin. Placenta and lymphoma sections, utilized as negative and positive controls, were run simultaneously.
IHC results were independently blindly interpreted by two pathologists (I.C. and P.Z.) to assess intrapersonal and interpersonal reproducibility.
IHC evaluation was performed through the nuclear (PAX5, BCL6, MUM1/IRF4, MYC, CyclinD1) and cytoplasmic membrane (CD10, BCL2, CD5, CD20, CD79a, CD3, CD4, CD8, CD2, CD30, ALK, Cytokeratin) positivity. For this purpose, 10 high-power fields were evaluated for each case and the proportion of cells with positive signals, rather than signal intensity, was quantified and assessed as positive or negative [1,2]. CD20, CD79a, PAX5, CD10, BCL6, MUM1/IRF4, CD3, CD68, CD5, CD2, CD4, CD8, CD138, CD30, ALK, CyclinD1 and Cytokeratin staining were considered positive when 30% or more of the cells showed antigen expression [1,2,17-19]. BCL2 staining was considered positive when expressed in over 50% of tumor cells [1,2,17-19]. MYC staining was considered positive when expressed in over 40% of tumor cells [1,2,17-19] A Ki-67 rate >50% of nuclei was considered a ‘‘high proliferation’’ rate [1,2]. Small or indeterminate cells were not considered in cell counting.
Immunoreactivity was evaluated without any knowledge of the patient’s survival or other clinical data. IHC results were merged with the histological features of PCBCL and nDLBCL.
Immunohistochemical classification
Hans’, Colomo’s and Muris’ algorithms, used for nDLBCL groups (GCB and non-GCB), were applied in order to assign the cell of origin (COO) to PCBCL [12,15-17]. In particular, PCBCL and nDLBCL were considered GCB when expressing CD10-/BCL6+/MUM1- or CD10+/BCL6±/MUM1- profiles by using the Hans’ algorithm [17], or MUM1-/CD10±/BCL6+ or MUM1-/CD10+/BCL6± profiles with the Colomo’s algorithm [18] or BCL2+/CD10±/MUM1- or BCL2-/CD10±/MUM1- with Muris’ algorithm [19]. PCBCL and nDLBCL were considered non-GCB when expressing the CD10-/BCL6±/MUM1+ profiles at Hans’ algorithm, or MUM1+/CD10-/BCL6± profiles at Colomo’s algorithm or BCL2+/CD10±/MUM1+ profiles at Muris’ algorithm.
Gene Expression Profiling
To assign the PCBCL to the GCB or activated B cell (ABC) groups, samples were analyzed with the Lymphoma/Leukemia Molecular Profiling Lymph2Cx assay, a digital gene expression (NanoString)–based test for COO assignment in FFPE tissue (FFPET).
Ten-micrometer scrolls of FFPET were cut with a surface area of ≥8 mm2 of each case tested by GEP. RNA was extracted using the Qiagen RNeasy FFPET kit (cat. N. 73504, Qiagen, Inc., Valencia, CA, USA), following the manufacturer’s instruction, after treatment with Qiagen Deparaffinization Solution (cat. N. 19093, Qiagen) and digital GEP was performed on 200 ng RNA using NanoString technology (Seattle, WA) with 23-Gene Signature for COO Classification Lymph2Cx (Nanostring Technologies, Seattle, WA, USA). GEP was performed on Affymetrix U133 plus 2.0 microarrays at Polo Tecnologico Pharmadiagen Srl
Laboratory (Pordenone, Italy). The data algorithm nCounter-based Lymph2Cx Assay Misclassification Rate was 2%. GEP analysed cases were blindly classified according to the COO assignment [6,7].
Gene expression profiling analysis
The 14 PCBCL and 14 nDLBCL samples were analysed using the RUO version of the GEP NanoString Lymphoma Subtyping Test (LST) algorithm to determine the COO molecular subtype of each sample. The LST algorithm measures the geometric mean of 5 housekeeping genes (HK geomean) to ensure RNA quality based on a pre-defined clinical quality control (QC) threshold of 128. An HK geomean value below 64 is deemed to be insufficient in terms of RNA quality to provide a subtyping result (subtype listed as N/A). A value between 64 and 128 is borderline quality since it meets previously published thresholds for RNA quality within clinical research studies [5,6], but does not meet the clinical QC threshold of 128 for individual patients. Samples that met the QC threshold were marked as “pass” (tab. 3).
Table 3
Sample stratification by IHC algorithms and GEP
Samples
(N.)
|
IHC Analysis
|
GEP
|
IHC Profiles
|
Hans
|
Colomo
|
Muris
|
1c
|
BCL2+/CD10-/BCL6+/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
2c
|
BCL2+/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
3c
|
BCL2-/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
4c
|
BCL2+/CD10-/BCL6-/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
Unclassified
|
5c
|
BCL2+/CD10-/BCL6-/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
6c
|
BCL2+/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
7c
|
BCL2+/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
8c
|
BCL2+/CD10-/BCL6-/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
9c
|
BCL2+/CD10-/BCL6-/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
10c
|
BCL2-/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
11c
|
BCL2+/CD10-/BCL6+/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
12c
|
BCL2+/CD10-/BCL6+/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
13c
|
BCL2-/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
14c
|
BCL2-/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
1n
|
BCL2+/CD10-/BCL6-/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
2n
|
BCL2+/CD10+/BCL6-/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
3n
|
BCL2+/CD10-/BCL6+/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
Unclassified
|
4n
|
BCL2+/CD10-/BCL6+/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
Unclassified
|
5n
|
BCL2+/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
6n
|
BCL2+/CD10-/BCL6+/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
7n
|
BCL2+/CD10-/BCL6+/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
8n
|
BCL2+/CD10-/BCL6-/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
9n
|
BCL2-/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
10n
|
BCL2-/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
11n
|
BCL2+/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
GCB
|
12n
|
BCL2+/CD10-/BCL6+/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
13n
|
BCL2-/CD10+/BCL6+/MUM1-
|
GCB
|
GCB
|
GCB
|
Unclassified
|
14n
|
BCL2+/CD10-/BCL6+/MUM1+
|
non-GCB
|
non-GCB
|
non-GCB
|
ABC
|
1c to 14c: primary cutaneous large, B-cell lymphoma cases; 1n to 14n: nodal DLBCL cases; ABC: Activated-B-Cell; GCB: Germinal Center-B-Cell; ABC: Activated-B-Cell; GCB: Germinal Center-B-Cell; IHC: immunohistochemistry; GEP: Gene Expression Profiling. |
Each sample meeting the QC threshold is reported as one of the three molecular subtypes GCB, or ABC or Unclassified within the equivocal zone. Subtypes are determined using a Linear Predictor Score (LPS), which is computed by summing the products of 15 weighted gene coefficients and the gene expression measurements and applying pre-defined thresholds [6,7]. Subtypes were ABC when LPS was higher or equal to 2433.5 (≥ 2433.5); GCB when LPS was lower or equal to 1907.8 (≤ 1907.8) and unclassified when LPS value was in equivocal zone (tab. 3).
t(14,18)(q32;q21) Polymerase Chain Reaction
To evaluate the t(14,18)(q32;q21) causing the BCL2-IGH rearrangements, genomic DNA was extracted by 8 sections (5–10 μm thick) using the Qiagen QIAamp DNA FFPE Tissue Kit (Qiagen, cat. n. 56404), following the manufacturer’s instructions, after treatment with Qiagen Deparaffinization Solution (Qiagen, cat. n. 19093). The kit combines the selective binding properties of a silica-based membrane by allowing the genomic DNA purification from FFPET sections without overnight incubation. Once paraffin was dissolved in xylene, samples were lysed under denaturing conditions with proteinase K and incubated at 90°C to reverse formalin cross-linking. The DNA was bound to the membrane and contaminants flowed through by centrifugation at 8000 rpm; DNA was then washed to remove the residual contaminants by centrifugation at 8000 rpm and then eluted from the membrane in 20-100 μl of Buffer ATE by centrifugation at 13200 rpm. In less than 30 minutes, the pure and concentrated DNA was ready for use in amplification reactions or for storage at –20°C.
The PCR analysis of t(14,18)(q32;q21) was carried out to evaluate BCL2-IGH rearrangement, according to Rambaldi A. et al. [20]. Briefly, the first round of amplification was in 50 μl of final volume of 1X GoTaq(R) Green Master Mix (2X; Promega, Madison, WI, USA, cat. n. M7123) using 100 ng of DNA with primers MBR for major breakpoint region, or mcr for minor cluster region and JH consensus region.
Samples were amplified on Veriti® Thermocycler (Applied Biosystems, Carlsbad, California, USA) with an initial denaturation at 95°C for 7 minutes, followed by either 27 cycles of denaturation for MBR or 30 cycles for the mcr. Each cycle was performed with 1 minute of denaturation at 94°C and 1 minute of annealing at 55°C for the MBR and 55°C for mcr amplifications and 1 minute of extension at 72°C. A nested PCR reaction was performed using 5 μl of 1:10 dilution of the first-round amplification product using oligonucleotide primers internal to the original primers, such as MBR nested, mcr nested and JH nested of which nucleotide sequences were described by Rambaldi A. et al. [20]. PCR reactions were performed according the following PCR temperature profile: denaturation for 7 minutes at 94°C, 30 cycles (1 minute at 94°C, 1 minute at 58°C and 1 minute at 72°C for each cycle) and final extension at 72°C for 10 minutes. A 25 μl aliquot of PCR product was verified on a 2% agarose gel containing ethidium bromide in Tris-borate electrophoresis buffer and visualized under UV light. Specifically, positive and negative PCR controls were processed, and samples were run in duplicate to ensure PCR reproducibility.
Data comparison
IHC data were compared to the GEP data to validate results and to one or more IHC algorithms to be used as GEP surrogate. Concordance percentage was calculated to determine the reproducibility between the GEP and IHC in PCBCL and nDLBCL. To evaluate the influence of biological, histological, immunohistochemical, or molecular data on the distribution of cases with GEP and IHC, ANOVA analysis was carried out with a significance of 5% and with two tails for independent data. Statistical analysis was performed using Graphpad Prism 8.0®. Different clinical and histological features were evaluated in discordant cases as possible cause of disagreement.