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Clonality testing of malignant lymphomas with the BIOMED-2 primers in a large cohort of 1969 primary and consultant biopsies
Pathology – Research and Practice 209 (2013) 495–502
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Original article
Clonality testing of malignant lymphomas with the BIOMED-2 primers in a large cohort of 1969 primary and consultant biopsies Sylvia Hartmann ∗,1 , Alexandra Helling 1 , Claudia Döring, Christoph Renné, Martin-Leo Hansmann Senckenberg Institute of Pathology, Goethe University, 60590 Frankfurt am Main, Germany
a r t i c l e
i n f o
Article history: Received 17 November 2012 Received in revised form 10 April 2013 Accepted 21 May 2013 Keywords: Clonality Non-Hodgkin lymphoma Hodgkin lymphoma Reactive lesions
a b s t r a c t The introduction of the BIOMED-2 primers allowed for reliable comparisons of clonality testing data of malignant lymphomas from different laboratories. This study undertook a retrospective analysis of a large cohort of cases; 1862 cases involved the immunoglobulin heavy chain locus (IGH VH –JH ), and 1527 cases involved the T cell receptor gamma locus (TCRG). We confirmed previously published clonality rates in various B cell, T cell, and Hodgkin lymphoma cases. In reactive lesions, clonality for the IGH locus was frequently accompanied by additional polyclonal background. Clonality for TCRG was found in a subgroup of diffuse large B cell lymphomas. On closer morphologic inspection, seven cases appeared to have arisen from an underlying peripheral T-cell lymphoma. Five cases with monoclonal TCRG rearrangements, originally diagnosed as Hodgkin lymphomas, were reclassified as T-cell lymphomas. TCRG clonality was very rarely only observed in Hodgkin lymphoma. In case of clear TCRG clonality a T-cell neoplasia must be ruled out on morphological grounds. By careful examination of the rearrangement patterns, including an assessment of a co-amplified polyclonal background, clonality testing provides a powerful tool which in concert with morphologic and immunohistochemical parameters can lead to a firm diagnosis. © 2013 Elsevier GmbH. All rights reserved.
Introduction Conventionally, the diagnosis of malignant lymphoma is based primarily on morphologic and immunohistochemical findings. Additional molecular studies, such as clonality testing, can be very helpful in certain cases. Whereas in rare cases malignant lymphomas, particularly of germinal center B cell origin, can present with polyclonal rearrangement patterns [1], reactive lesions can mimic lymphoma showing clonal peaks in gene scanning [2]. Since the BIOMED-2 primers for the amplification of B cell and T cell receptor gene rearrangements were established in 2003 [3], they have been extensively validated in formalin-fixed, paraffinembedded (FFPE) tissues [4,5], and results of analyses performed in different institutions are highly comparable. We therefore retrospectively analyzed the results of clonality testing in our laboratory in a large cohort of 1969 formalin-fixed paraffin embedded cases. Knowledge of the clonality results has been used in making the original diagnosis in individual cases. For a variety of
∗ Corresponding author at: Senckenberg Institute of Pathology, University of Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. Tel.: +49 69 63014284; fax: +49 69 63015241. E-mail address: [email protected] (S. Hartmann). 1 These authors contributed equally to this work. 0344-0338/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2013.05.004
reasons, there were many cases in which a B cell lymphoma was suspected, but we also tested T cell clonality, and there were cases in which a T cell lymphoma was suspected, but we also tested B cell clonality. Additional insights were obtained by retrospective analysis of the presence of a polyclonal background in cases that exhibited B cell clonality, a feature that was not examined during the original diagnostic procedures. Thus, this study provides information that is important to laboratories employing clonality testing in routine practice. The study also demonstrates that clonality testing complements primary morphological and immunohistochemical findings and can help resolve differential diagnostic questions.
Materials and methods Tissue samples We analyzed all samples diagnosed at the Senckenberg Institute of Pathology between April 2005 and May 2009, which had been tested for clonality with suspicion of lymphoma. The study includes a broad spectrum of lymphoma and reactive cases, which were sent for primary diagnosis or as consultant cases to the department. Consent of the local ethics committee was obtained for the study. A clonality test was
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Table 1 IGH VH –JH rearrangements in the evaluable cases of different entities. Entity
Number of evaluable cases
Number of clonal cases without any polyclonal background (%)
B cell lymphomas (n = 763)
MZL FL DLBCL LPL MCL B-CLL PLC PTLD BL PMBCL
260 198 187 38 27 22 13 11 5 2
228 (88) 159 (80) 150 (80) 36 (95) 27 (100) 19 (86) 11 (85) 9 (82) 5 (100) 2 (100)
Hodgkin (n = 68)
cHL NLPHL
54 14
10 (19) 0 (0)
7 (13) 4 (29)
T cell neoplasias (n = 169)
AITL PTCL NOS ALCL MF NKTL EATL LyP
61 42 21 20 6 5 14
15 (25) 6 (14) 5 (24) 3 (15) 0 (0) 1 (20) 0 (0)
1 (2) 2 (5) 1 (5) 1 (5) 0 (0) 0 (0) 3 (21)
Reactive (n = 401)
LA unspecified Cutaneous PL Castleman’s Kikuchi
345 40 10 6
9 (3) 6 (15) 0 (0) 0 (0)
Number of clonal cases with polyclonal background (%) 9 (2) 6 (3) 6 (3) 0 (0) 0 (0) 1 (4) 0 (0) 0 (0) 0 (0) 0 (0)
27 (8) 1 (3) 0 (0) 0 (0)
Number of oligoclonal cases (%)
Number of polyclonal cases (%)
0 (0) 1 (1) 2 (1) 0 (0) 0 (0) 2 (10) 0 (0) 1 (9) 0 (0) 0 (0)
23 (10) 32 (16) 29 (16) 2 (5) 0 (0) 0 (0) 2 (15) 1 (9) 0 (0) 0 (0)
17 (32) 4 (29)
0 (0) 0 (0)
37 (69) 10 (71)
16 (26) 8 (19) 6 (29) 4 (20) 0 (0) 1 (20) 3 (21)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
45 (74) 34 (81) 15 (71) 16 (80) 6 (100) 4 (80) 11 (79)
36 (10) 7 (18) 0 (0) 0 (0)
1 (0) 0 (0) 0 (0) 0 (0)
308 (89) 33 (83) 10 (100) 6 (100)
Number of all clonal cases (%) 237 (90) 165 (83) 156 (83) 36 (95) 27 (100) 20 (90) 11 (85) 9 (82) 5 (100) 2 (100)
MZL, marginal zone lymphoma; FL, follicular lymphoma; DLBCL, diffuse large B cell lymphoma; LPL, lymphoplasmocytic lymphoma; MCL, mantle cell lymphoma; B-CLL, lymphocytic lymphoma/leukemia; PLC, plasmacytoma; PTLD, posttransplant lymphoproliferative disorder; BL, Burkitt lymphoma; PMBCL, primary mediastinal B cell lymphoma; cHL, classical Hodgkin lymphoma; NLPHL, nodular lymphocyte predominant Hodgkin lymphoma; AITL, angioimmunoblastic T cell lymphoma; PTCL NOS, peripheral T cell lymphoma not otherwise specified; ALCL, anaplastic large cell lymphoma; MF, mycosis fungoides; NKTL, NK/T cell lymphoma; EATL, enteropathy associated T cell lymphoma; LA unspecified, unspecified lymphadenitis; Cutaneous PL, cutaneous pseudolymphoma; LyP, lymphomatoid Papulosis; Castleman’s, Castleman’s disease; Kikuchi, Kikuchi Lymphadenitis.
ordered by the diagnostic pathologist at the initial review of HE and Giemsa slides in all cases that did not show a classic morphological pattern. Final diagnoses were based on morphological and immunohistochemical findings in concert with results of clonality testing.
DNA extraction, multiplex PCR amplification and PCR product analysis Genomic DNA was extracted from 60 to 120 m of paraffinembedded tissue after deparaffinization using the QiaAmp Mini Kit (Qiagen, Hilden, Germany). Samples were divided into three groups based on DNA concentrations: <10 ng/l, 10–50 ng/l and >50 ng/l. The BIOMED-2 multiplex PCRs for the immunoglobulin heavy chain (IGH) VH –JH rearrangements (tubes A–C, IdentiClone, Invivoscribe Technologies, La Ciotat, France), as well as the multiplex reaction for T cell receptor gamma (TCRG) rearrangements (tubes A and B, IdentiClone), were performed as described previously [3] with 45 cycles. All samples were run in duplicates. Positive (clonal) and negative (polyclonal) controls in addition to a blank control (H2 O) were included in all cases. Gene scanning was carried out on an ABI-Prism 3100 Sequencer (Life Technologies, Darmstadt, Germany) using the Gene Scan Analysis Software 3.7. Cases were tested for IGH and/or TCRG rearrangements. The results were scored “clonal” if either the IGH or TCRG multiplex PCRs yielded a reproducible clonal product at the same position in both replicates. If apparent clonal peaks were of different fragment lengths in the two replicates (pseudoclonality), the analysis was scored not evaluable. The analysis was scored “clonal with polyclonal background” if the reactions showed a reproducible clonal product at least 3 times the intensity of the third largest peak observed.
Results Cases and diagnoses Of the 1969 cases tested for clonality, 1862 cases were tested for IGH and 1527 for TCRG clonality. Of these, 851 cases (43%) represented B cell lymphomas, 296 cases (15%) were diagnosed as T cell neoplasias, 89 cases were Hodgkin lymphomas (5%), 575 cases (29%) were finally classified as reactive lesions, and in 171 cases (9%), a non-lymphoid neoplasm was diagnosed or no definite diagnosis could be established. Detailed information about cases and diagnoses is found in Tables 1 and 2. Most specimens represented lymph nodes (511 cases). While B cell lymphomas dominated in all localizations except the skin (61–92%), T cell neoplasias (59%) were more frequently observed in skin than B cell lymphomas (41%). Interpretability of clonality analyses To assess the influence of the DNA concentration, we performed clonality analyses for Frameworks II and III of the IGH locus with cell line DNA (Raji cell line, a Burkitt lymphoma cell line) in different dilutions (starting from 446 ng/dl, 1:1, 1:3, 1:9, 1:27, 1:81, 1:243 and 1:729). We chose Frameworks II and III, since they are more frequently evaluable in primary samples than Framework I due to a shorter length of the PCR products. Optimal results for both Framework PCRs were obtained if the DNA concentration was ≥50 ng/dl (1:9 dilution), but reproducible peaks of the expected length could still be detected in both Framework reactions at a dilution of 1:81 (<10 ng/dl). To determine the influence of the DNA concentration on the interpretability of the clonality results, the samples were grouped according to DNA concentration: <10 ng/l, 10–50 ng/l and >50 ng/l in order to determine the influence of the DNA
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Table 2 TCRG rearrangements in the evaluable cases in different entities. Number of clonal cases without any polyclonal background (%)
Entity
Number of evaluable cases
B cell lymphomas (n = 448)
MZL FL DLBCL LPL MCL B-CLL PLC PTLD
124 110 147 26 15 15 5 6
Hodgkin (n = 70)
cHL NLPHL
58 12
1 (2) 1 (8)
T cell neoplasias (n = 266)
AITL PTCL NOS ALCL MF NKTL EATL SPLTL LyP
66 82 41 35 9 5 5 23
46 (70) 63 (77) 34 (83) 18 (51) 1 (11) 4 (80) 2 (40) 12 (52)
Reactive (n = 339)
LA unspecified Cutaneous PL Castleman’s Parapsoriasis Kikuchi
287 36 6 5 5
9 (3) 2 (6) 0 (0) 2 (40) 0 (0)
3 (3) 4 (4) 15 (10) 2 (8) 0 (0) 0 (0) 0 (0) 0 (0)
Number of clonal cases with polyclonal background (%)
Number of all clonal cases (%)
Number of oligoclonal cases (%)
Number of polyclonal cases (%)
31 (24) 26 (24) 38 (26) 4 (16) 4 (27) 3 (20) 3 (60) 1 (17)
34 (27) 30 (27) 53 (36) 6 (23) 4 (27) 3 (20) 3 (60) 1 (17)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
90 (73) 80 (73) 94 (64) 20 (76) 11 (73) 12 (80) 2 (40) 5 (83)
9 (16) 2 (17)
10 (17) 3 (25)
1 (2) 0 (0)
47 (81) 9 (75)
15 (23) 9 (11) 4 (10) 15 (43) 3 (33) 1 (20) 2 (40) 9 (39)
61 (92) 72 (88) 38 (93) 33 (94) 4 (44) 5 (100) 4 (80) 21 (91)
0 (0) 0 (0) 0 (0) 1 (3) 0 (0) 0 (0) 0 (0) 0 (0)
5 (8) 10 (12) 3 (7) 1 (3) 5 (56) 0 (0) 1 (20) 2 (9)
69 (24) 7 (19) 0 (0) 1 (20) 0 (0)
78 (27) 9 (25) 0 (0) 3 (60) 0 (0)
3 (1) 0 (0) 0 (0) 0 (0) 0 (0)
206 (72) 27 (75) 6 (100) 2 (40) 5 (100)
MZL, marginal zone lymphoma; FL, follicular lymphoma; DLBCL, diffuse large B cell lymphoma; LPL, lymphoplasmocytic lymphoma; MCL, mantle cell lymphoma; B-CLL, lymphocytic lymphoma/leukemia; PLC, plasmacytoma; PTLD, posttransplant lymphoproliferative disorder; BL, Burkitt lymphoma; cHL, classical Hodgkin lymphoma; NLPHL, nodular lymphocyte predominant Hodgkin lymphoma; AITL, angioimmunoblastic T cell lymphoma; PTCL NOS, peripheral T cell lymphoma not otherwise specified; ALCL, anaplastic large cell lymphoma; MF, mycosis fungoides; NKTL, NK/T cell lymphoma; EATL, enteropathy associated T cell lymphoma; SPLTL, subcutaneous panniculitis-like T cell lymphoma; LA unspecified, unspecified lymphadenitis; Cutaneous PL, cutaneous pseudolymphoma; LyP, lymphomatoid Papulosis; Castleman’s, Castleman’s disease; Kikuchi, Kikuchi Lymphadenitis.
yield non-interpretable results (7 and 5 samples for IGH and TCRG, respectively) compared to samples with >500 cells/section (2 and 3 samples for IGH and TCRG, respectively, Suppl. Tables 1 and 2). Comparably, the number of samples with monoclonal results was higher if the content of lymphoid cells/section was above 500 (16 and 9 samples for IGH and TCRG, respectively) compared to samples with <100 cells/section (2 and 1 sample(s) for IGH and TCRG, respectively). However, eleven B cell lymphoma samples with a content of <500 clonal cells/section still yielded reproducible clonal peaks in at least one Framework reaction.
Clonality in B cell lymphomas Fig. 1. Interpretability of clonality results in different assays. Samples are sorted by DNA concentrations (<10 ng/l, 10–50 ng/l, >50 ng/l). Percentages of evaluable clonality assays in relation to all cases tested are displayed. Frameworks I–III (tubes A–C) of the IGH VH –JH rearrangements (n = 1862), TCRG T cell receptor gamma (n = 1527). **p < 0.01, Chi-Square Test.
concentration on the interpretability of the clonality results. The interpretability of the results for the three regions Framework I–III of IGH VH –JH , as well as TCRG, was significantly dependent on the DNA concentrations (Fig. 1, Chi-Square Test p < 0.01). Therefore, in all the reactions, samples were more likely to be evaluable when the DNA concentration was above a critical threshold of 50 ng/l. Nevertheless, overall 64% of IGH and 56% of TCRG cases with a DNA concentration <10 ng/l still yielded evaluable results. Since the interpretability of the clonality assays does not only depend on the DNA concentration but also on the number of lymphocytes in the tissue, we assessed the number of B and T cells in the histologic section for all samples with a DNA content <10 ng/dl. Samples with a content of <100 cells/section of the respective lineage had a higher probability to
Mature B cell lymphomas showed clonal IGH VH –JH rearrangements in at least 83% of the evaluable cases. The highest frequencies of clonal IGH rearrangement rates (100%) were found in mantle cell lymphoma, mediastinal B cell lymphoma, and Burkitt lymphoma. Clonal IGH rearrangements were found in 83% of typical germinal center-derived follicular lymphoma (FL) and germinal center or post-germinal center-derived diffuse large B cell lymphoma (DLBCL). Monoclonal IGH rearrangements were seen in 85–95% of other “low grade” B cell lymphomas, including lymphocytic lymphoma (B-CLL), marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma, and plasmacytoma, which are in most cases post-germinal center-derived and have predominantly undergone the somatic hypermutation process (Table 1). Clonal rearrangements of the TCRG locus were most frequently found in DLBCL (36% of 147 evaluable DLBCL). All other B cell lymphoma entities (groups consisting of at least 15 cases) showed clonal rearrangements of the TCRG in ≤27% of the cases. All DLBCL with clonal TCRG rearrangements were retrospectively reviewed. At close inspection, among the 15 cases with a monoclonal TCRG rearrangement pattern without polyclonal background, seven cases were identified
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Fig. 2. Pleomorphic monoclonal T cell proliferation in a diffuse large B cell lymphoma, suggesting an underlying peripheral T cell lymphoma. (a) HE, 200×. (b) Sheets of B cell blasts. CD20-immunostaining, 200× (CD20, 1:1000, DAKO, Glostrup, Denmark). (c) CD3-immunostaining highlighting large pleomorphic T cells (arrows), 400× (CD3, 1:200, Novocastra, Newcastle upon Tyne, UK). (d) Monoclonal rearrangement of TCRG.
with either remarkably pleomorphic T cells or and aberrant T cell immunophenotype (Figs. 2 and 3), suggesting a true monoclonal T cell proliferation. Only two cases presented an epithelioid cell-rich background, consistent with an underlying angioimmunoblastic T cell lymphoma (AITL). Age distribution did not differ between DLBCL patients with or without clonal TCRG rearrangement (mean 62 vs. 61 years, respectively). Among the cases exhibiting a monoclonal TCRG peak with additional polyclonal background, no further evidence of a T cell neoplasia was found. Clonality in T cell neoplasias The various T cell lymphoma entities exhibited clonal rearrangements of the TCRG in the majority of cases (Table 2). Interestingly, AITL and mycosis fungoides (MF) showed the highest percentage of TCRG rearrangement (94% each), whereas only 88% of peripheral T cell lymphomas not otherwise specified (PTCL NOS) showed clonal rearrangements. Clonal VH –JH rearrangements of the IGH locus were rarely found in PTCL NOS and MF (16% and 20%, respectively). However, 29% of anaplastic large cell lymphomas and 25% of AITL showed clonal IGH rearrangements. Clonality in Hodgkin lymphoma Cases of classical and nodular lymphocyte predominant HL (NLPHL) exhibited similar frequencies of clonal rearrangements of the IGH locus (32% and 29%, respectively) and TCRG locus (17%
and 25%, respectively). All HL with clonal rearrangements of either IGH or TCRG were retrospectively reviewed. In cases of classical HL cases with a clonal IGH rearrangement, high numbers of Hodgkinand Reed–Sternberg cells were usually observed. In NLPHL clonal IGH rearrangements could not be explained by high numbers of tumor cells; however, in these cases, the clonal peak was always associated with a polyclonal background. Monoclonal TCRG rearrangements without a polyclonal background were only rarely observed in classical HL and NLPHL (one case each). Three cases originally classified as classical HL (with EBV-infected Hodgkin- and Reed–Sternberg cells) and two NLPHL with monoclonal TCRG rearrangement were reclassified as PTCL NOS or AILT after the review and were included in those categories for the remainder of the study.
Clonality in reactive lesions In a series of 598 reactive lesions, 459 were tested for rearrangements of the IGH locus and 362 for TCRG rearrangements. The vast majority of reactive lesions showed polyclonal patterns for the IGH locus (368 of 415 evaluable cases, 89%), as well as for the TCRG (248 of 362 evaluable cases, 69%). Monoclonal rearrangements of the IGH locus occurred in 36 cases (10%); of these, there were 9 cases in which a monoclonal product without polyclonal background was detected in at least one framework (2% of all reactive lesions). Three of these cases were lymph nodes from children showing massive follicular hyperplasia. Clinical follow up data (1
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Fig. 3. Monoclonal T cell proliferation with an aberrant immunophenotype in a diffuse large B cell lymphoma, suggesting an underlying peripheral T cell lymphoma. (a) HE, 200×. (b) Nodular accumulation of B cell blasts in CD20 immunostaining, 100×. (c) CD3-positive T cells surrounding the nodules of B blasts, 100×. (d) CD10-immunostaining highlighting the T cells, while B cell blasts remain negative, 100× (CD10, 1:100, Novocastra). (e) Ki67-immunostaining showing an elevated proliferation rate, particularly in the B cell blasts, 100× (Ki67, 1:200, DAKO). (f) Monoclonal rearrangement of TCRG.
month–4 years) were available for four additional patients. None of these patients developed a lymphoma. However, since pediatric follicular lymphoma often presents with a localized stage, can be BCL2-negative [6], and can be cured by simple lymphadenectomy, we cannot completely rule out the infiltration by pediatric follicular lymphoma in the three children. The majority of reactive lesions with clonal rearrangement patterns (27/36 cases, 75%) presented a polyclonal background, and in 20 of these cases (56%), the clonal amplificate was restricted to one of the three VH rearrangement PCRs (Fig. 4). In contrast, B cell lymphomas more frequently exhibited a clear monoclonal rearrangement in at least two VH PCR assays without a concomitant polyclonal background (444 cases, 66%,
p < 0.05, Fishers Exact Test, Fig. 4). Compared to polyclonal reactive lesions, reactive lesions with clonal IGH rearrangements were more likely, albeit not significantly, to be localized in the gastrointestinal tract (11% vs. 4%) or respiratory tract (36% vs. 20%). Clonal rearrangements of the TCRG in reactive lesions were observed approximately at the same frequency as clonal rearrangements of the IGH locus (90 of 339 cases, 27%). Clear monoclonal products without any polyclonal background were found in 9 nonspecific reactive lesions (3% of all reactive lesions). Two of these patients were HIV-positive, and one was hepatitis C-positive. Clinical follow up data (5 months–7 years) were available in three patients. None of the patients developed a lymphoma. Interestingly, the
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Fig. 4. Pattern distribution of clonally rearranged IGH framework regions in B cell lymphomas and reactive lesions. (a) Typical monoclonal product without polyclonal background in Framework PCR II (tube B of IGH) in a B cell lymphoma. (b) Typical pattern of monoclonal product with polyclonal background in Framework PCR II (tube B of IGH) in a non-specific lymphadenitis. (c) B cell lymphomas and reactive lesions with clonally rearranged IGH loci, sorted by the number of clonally rearranged framework PCRs and the presence of additional polyclonal background. **p < 0.01, *p < 0.05, Fisher’s Exact Test.
frequency of clonal rearrangements of the IGH locus or the TCRG locus in cutaneous pseudolymphoma (18% and 25%, respectively) was similar to the frequencies of clonal IGH and TCRG rearrangements observed in nonspecific reactive lesions of other localizations (15% and 27%, respectively). Discussion Clonality testing is performed in many laboratories using the BIOMED-2 primers [3], and results are highly comparable between laboratories. We evaluated a large cohort of consultant cases and primary diagnostic samples tested for clonality. Selection criteria for clonality testing were either to confirm the conventional diagnosis or to resolve differential diagnostic questions. The data may be biased, since the final diagnosis included the results of clonality testing; however, the overall results match very well with the findings observed by others: in the present study, clonal IGH VH –H rearrangements were observed in 83% of each the FL and DLBCL in the evaluable cases; in the study by Evans et al. [1], 84% and 79% of the FL and DLBCL, respectively, showed clonal IGH VH –H rearrangements. Other investigators observed lower percentages of IGH rearrangements: in FL for example, frequencies ranged from 31% to 80% when only the IGH approach was applied [7–10]. These differences may be due to different technical reasons. We are also aware that because of somatic hypermutation of the IGH locus [3], the sensitivity of clonality detection can be dramatically increased by the light chain assays [1,7,8,10].
In line with previous investigations [10], we found the DNA concentration to be an important factor for the interpretability of the data. Nonetheless, still 53% of samples with DNA contents <10 ng/l showed evaluable results for VH -FR3–JH . Pseudoclonal results, likely to be obtained in samples containing only small numbers of lymphocytes [11], were scored not evaluable. A content of >500 lymphoid cells per section of the respective lineage proved to be critical for reliable PCR results. However, in rare cases of B cell lymphomas, clonal patterns for IGH could be demonstrated even if the content of clonal cells per section was <500. This indicates that the number of cells required for an interpretable result is less in a monoclonal than in a polyclonal population. Therefore, a clonality test always seems to be worth trying. However, due to the low number of samples with a DNA content below 10 ng/dl, these results should be interpreted with caution. Interestingly, an additional clonal TCRG rearrangement was most frequently detected in DLBCL (36%, Table 2). In the study by Evans et al. [1], clonal rearrangements of the TCRG were likewise most frequently observed in DLBCL, extranodal MALT lymphoma, and B-CLL (15–18%). In the present study, seven of these cases showed a T cell infiltrate either with pleomorphism or with an aberrant immunophenotype, suggesting an underlying T cell lymphoma which was overgrown and effaced by an emerging large B cell lymphoma. In all other cases with clonal TCRG rearrangements, no morphologic correlate of the T cell clone could be identified. Therefore, DLBCL with clonal TCRG rearrangements can be divided into two groups: on the one hand DLBCL arising in
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previously existing T cell lymphoma, particularly AITL or PTCL NOS [12–14]. On the other hand DLBCL developing on a background of an impaired immune control for other reasons, as can be observed in the elderly or in immunocompromised individuals. Dojcinov et al. [15] observed restricted or monoclonal T cell repertoires in 30% of EBV-positive DLBCL. A similar issue was addressed in a study by Ibrahim et al. [16], who found clonal T cell expansions in 50% of PTLD. Hence, the number of DLBCL arising in an immunocompromised background with impaired T cell control of the malignant clone may be higher than expected. Another explanation for clonal TCRG rearrangements in DLBCL may be a very low T cell content in the tissue of some cases. We observed similar rates of clonal TCRG rearrangements in PTCL NOS, ALCL, and AITL as noted in previous reports [17,18]. For AITL, this is surprising, as previous studies [19] showed that the neoplastic population may be a minority in AITL. B cell clonality (IGH) in T cell lymphomas was detected in the present study in 19–29% (Table 1), which is in line with previously reported rates of 30% in PTCL NOS [20], 9–11% in ALCL [18], and 19% in AITL [17]. Emerging B cell lymphomas have been described in many T cell lymphoma subsets [12,13,18,21], and monoclonal IGH rearrangements may be indicative of an early B cell expansion due to the impaired control of the immune system in the setting of a T cell neoplasia. This may particularly be the case for EBV-infected B cell clones. We detected clonality for IGH in approximately 1/3 of the HL cases. In 19% of classical HL, clonal products lacking polyclonal background were found, which could morphologically be explained by an abundant tumor cell infiltrate. IGH clonality in classical HL was previously reported in 16% [22], 21% [23], 40% [24], and 67% [25] of the cases. In all NLPHL showing clonal IGH rearrangements, the clonal product was found to be associated with a polyclonal background infiltrate, reflecting an abundance of reactive B cells in the infiltrate. Interestingly, of four original classical HL with a monoclonal TCRG product, after histopathologic review, three cases had to be reclassified as T cell lymphomas (2 as PTCL NOS with Hodgkin- and Reed–Sternberg cells and 1 AITL), and two NLPHL were reclassified as PTCL NOS. There are contradictory data regarding clonality of rosetting T cells around LP cells [26,27]. However, the number of cases investigated by microdissection of rosetting T cells is very low. Previously described atypical T cells occurring in NLPHL were reported to be polyclonal [28]. Therefore, HL with a clear monoclonal TCR rearrangement very likely represents particular PTCL with a “Hodgkin-like” morphologic presentation [29]. Among reactive lesions, clonal TCRG rearrangements with a polyclonal background infiltrate were quite frequent (23%). This phenomenon is very likely due to an antigen-driven clonal selection. Clonal rearrangements lacking a polyclonal background were rarely found among reactive lesions (3%) compared to nodal T cell lymphomas (where >70% lacked a polyclonal background). For the IGH locus, clonal products were even rarer; only 2% of all reactive lesions showed a clonal product for IGH without a polyclonal background infiltrate. Similar results were obtained by Langerak et al. who detected a clear monoclonal product for one Immunoglobulin or TCR target in 10% of reactive samples [2]. In our analysis, reactive lesions with clonal IGH patterns in most cases included an additional admixture of polyclonal products (8%), and clonality was mostly observed only in one or two Framework PCRs only. This is likely due to a preferential PCR amplification of one clone within a polyclonal infiltrate, which is more likely to happen in only one or two Framework PCRs than in all three reactions. Additionally, the localization of the diagnostic biopsy should be taken into account. We more often observed clonal IGH amplificates in reactive lesions from extranodal localizations, like gastrointestinal or respiratory tract. One reason may be the extensive exposure of mucosal
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surfaces to antigens that might stimulate the proliferation of specific B cell clones [30,31]. In summary, the present study shows that the BIOMED-2 primers represent a powerful diagnostic tool for assessing the clonality of B and T cells in reactive and neoplastic conditions. By careful examination of the rearrangement patterns, including an assessment of a co-amplified polyclonal background, an assessment of clonal rearrangements among multiple PCR reactions, as well as considering the localization of the biopsy, important information can be obtained, which in concert with morphologic and immunohistochemical parameters can lead to a firm diagnosis. Conflict of interest The authors report no potential conflict of interest. Acknowledgements The authors would like to thank Susanne Hansen for her excellent technical assistance. The authors thank Prof. Ralf Küppers, Essen, Germany, for his helpful discussions and comments on the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prp.2013.05.004. References [1] P.A. Evans, C. Pott, P.J. Groenen, G. Salles, F. Davi, F. Berger, et al., Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936, Leukemia 21 (2007) 207–214. [2] A.W. Langerak, T.J. Molina, F.L. Lavender, D. Pearson, T. Flohr, C. Sambade, et al., Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936, Leukemia 21 (2007) 222–229. [3] J.J. van Dongen, A.W. Langerak, M. Bruggemann, P.A. Evans, M. Hummel, F.L. Lavender, et al., Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936, Leukemia 17 (2003) 2257–2317. [4] R.F. McClure, P. Kaur, E. Pagel, P.D. Ouillette, C.E. Holtegaard, C.L. Treptow, et al., Validation of immunoglobulin gene rearrangement detection by PCR using commercially available BIOMED-2 primers, Leukemia 20 (2006) 176–179. [5] H. Liu, A.J. Bench, C.M. Bacon, K. Payne, Y. Huang, M.A. Scott, et al., A practical strategy for the routine use of BIOMED-2 PCR assays for detection of B- and T-cell clonality in diagnostic haematopathology, Br. J. Haematol. 138 (2007) 31–43. [6] I. Oschlies, I. Salaverria, F. Mahn, A. Meinhardt, M. Zimmermann, W. Woessmann, et al., Pediatric follicular lymphoma – a clinico-pathological study of a population-based series of patients treated within the Non-Hodgkin’s Lymphoma–Berlin–Frankfurt–Munster (NHL-BFM) multicenter trials, Haematologica 95 (2010) 253–259. [7] A.M. Halldorsdottir, B.A. Zehnbauer, W.R. Burack, Application of BIOMED2 clonality assays to formalin-fixed paraffin embedded follicular lymphoma specimens: superior performance of the IGK assays compared to IGH for suboptimal specimens, Leuk. Lymphoma 48 (2007) 1338–1343. [8] K. Payne, P. Wright, J.W. Grant, Y. Huang, R. Hamoudi, C.M. Bacon, et al., BIOMED-2 PCR assays for IGK gene rearrangements are essential for B-cell clonality analysis in follicular lymphoma, Br. J. Haematol. 155 (2011) 84–92. [9] Y.L. Chen, I.J. Su, H.Y. Cheng, K.C. Chang, C.C. Lu, N.H. Chow, et al., BIOMED-2 protocols to detect clonal immunoglobulin and T-cell receptor gene rearrangements in B- and T-cell lymphomas in southern Taiwan, Leuk. Lymphoma 51 (2010) 650–655. [10] E. Berget, L. Helgeland, A. Molven, O.K. Vintermyr, Detection of clonality in follicular lymphoma using formalin-fixed, paraffin-embedded tissue samples and BIOMED-2 immunoglobulin primers, J. Clin. Pathol. 64 (2011) 37–41. [11] A. Boer, R. Tirumalae, M. Bresch, T.M. Falk, Pseudoclonality in cutaneous pseudolymphomas: a pitfall in interpretation of rearrangement studies, Br. J. Dermatol. 159 (2008) 394–402. [12] K. Willenbrock, A. Brauninger, M.L. Hansmann, Frequent occurrence of Bcell lymphomas in angioimmunoblastic T-cell lymphoma and proliferation of Epstein–Barr virus-infected cells in early cases, Br. J. Haematol. 138 (2007) 733–739.
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Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original article
Clonality testing of malignant lymphomas with the BIOMED-2 primers in a large cohort of 1969 primary and consultant biopsies Sylvia Hartmann ∗,1 , Alexandra Helling 1 , Claudia Döring, Christoph Renné, Martin-Leo Hansmann Senckenberg Institute of Pathology, Goethe University, 60590 Frankfurt am Main, Germany
a r t i c l e
i n f o
Article history: Received 17 November 2012 Received in revised form 10 April 2013 Accepted 21 May 2013 Keywords: Clonality Non-Hodgkin lymphoma Hodgkin lymphoma Reactive lesions
a b s t r a c t The introduction of the BIOMED-2 primers allowed for reliable comparisons of clonality testing data of malignant lymphomas from different laboratories. This study undertook a retrospective analysis of a large cohort of cases; 1862 cases involved the immunoglobulin heavy chain locus (IGH VH –JH ), and 1527 cases involved the T cell receptor gamma locus (TCRG). We confirmed previously published clonality rates in various B cell, T cell, and Hodgkin lymphoma cases. In reactive lesions, clonality for the IGH locus was frequently accompanied by additional polyclonal background. Clonality for TCRG was found in a subgroup of diffuse large B cell lymphomas. On closer morphologic inspection, seven cases appeared to have arisen from an underlying peripheral T-cell lymphoma. Five cases with monoclonal TCRG rearrangements, originally diagnosed as Hodgkin lymphomas, were reclassified as T-cell lymphomas. TCRG clonality was very rarely only observed in Hodgkin lymphoma. In case of clear TCRG clonality a T-cell neoplasia must be ruled out on morphological grounds. By careful examination of the rearrangement patterns, including an assessment of a co-amplified polyclonal background, clonality testing provides a powerful tool which in concert with morphologic and immunohistochemical parameters can lead to a firm diagnosis. © 2013 Elsevier GmbH. All rights reserved.
Introduction Conventionally, the diagnosis of malignant lymphoma is based primarily on morphologic and immunohistochemical findings. Additional molecular studies, such as clonality testing, can be very helpful in certain cases. Whereas in rare cases malignant lymphomas, particularly of germinal center B cell origin, can present with polyclonal rearrangement patterns [1], reactive lesions can mimic lymphoma showing clonal peaks in gene scanning [2]. Since the BIOMED-2 primers for the amplification of B cell and T cell receptor gene rearrangements were established in 2003 [3], they have been extensively validated in formalin-fixed, paraffinembedded (FFPE) tissues [4,5], and results of analyses performed in different institutions are highly comparable. We therefore retrospectively analyzed the results of clonality testing in our laboratory in a large cohort of 1969 formalin-fixed paraffin embedded cases. Knowledge of the clonality results has been used in making the original diagnosis in individual cases. For a variety of
∗ Corresponding author at: Senckenberg Institute of Pathology, University of Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. Tel.: +49 69 63014284; fax: +49 69 63015241. E-mail address: [email protected] (S. Hartmann). 1 These authors contributed equally to this work. 0344-0338/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2013.05.004
reasons, there were many cases in which a B cell lymphoma was suspected, but we also tested T cell clonality, and there were cases in which a T cell lymphoma was suspected, but we also tested B cell clonality. Additional insights were obtained by retrospective analysis of the presence of a polyclonal background in cases that exhibited B cell clonality, a feature that was not examined during the original diagnostic procedures. Thus, this study provides information that is important to laboratories employing clonality testing in routine practice. The study also demonstrates that clonality testing complements primary morphological and immunohistochemical findings and can help resolve differential diagnostic questions.
Materials and methods Tissue samples We analyzed all samples diagnosed at the Senckenberg Institute of Pathology between April 2005 and May 2009, which had been tested for clonality with suspicion of lymphoma. The study includes a broad spectrum of lymphoma and reactive cases, which were sent for primary diagnosis or as consultant cases to the department. Consent of the local ethics committee was obtained for the study. A clonality test was
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Table 1 IGH VH –JH rearrangements in the evaluable cases of different entities. Entity
Number of evaluable cases
Number of clonal cases without any polyclonal background (%)
B cell lymphomas (n = 763)
MZL FL DLBCL LPL MCL B-CLL PLC PTLD BL PMBCL
260 198 187 38 27 22 13 11 5 2
228 (88) 159 (80) 150 (80) 36 (95) 27 (100) 19 (86) 11 (85) 9 (82) 5 (100) 2 (100)
Hodgkin (n = 68)
cHL NLPHL
54 14
10 (19) 0 (0)
7 (13) 4 (29)
T cell neoplasias (n = 169)
AITL PTCL NOS ALCL MF NKTL EATL LyP
61 42 21 20 6 5 14
15 (25) 6 (14) 5 (24) 3 (15) 0 (0) 1 (20) 0 (0)
1 (2) 2 (5) 1 (5) 1 (5) 0 (0) 0 (0) 3 (21)
Reactive (n = 401)
LA unspecified Cutaneous PL Castleman’s Kikuchi
345 40 10 6
9 (3) 6 (15) 0 (0) 0 (0)
Number of clonal cases with polyclonal background (%) 9 (2) 6 (3) 6 (3) 0 (0) 0 (0) 1 (4) 0 (0) 0 (0) 0 (0) 0 (0)
27 (8) 1 (3) 0 (0) 0 (0)
Number of oligoclonal cases (%)
Number of polyclonal cases (%)
0 (0) 1 (1) 2 (1) 0 (0) 0 (0) 2 (10) 0 (0) 1 (9) 0 (0) 0 (0)
23 (10) 32 (16) 29 (16) 2 (5) 0 (0) 0 (0) 2 (15) 1 (9) 0 (0) 0 (0)
17 (32) 4 (29)
0 (0) 0 (0)
37 (69) 10 (71)
16 (26) 8 (19) 6 (29) 4 (20) 0 (0) 1 (20) 3 (21)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
45 (74) 34 (81) 15 (71) 16 (80) 6 (100) 4 (80) 11 (79)
36 (10) 7 (18) 0 (0) 0 (0)
1 (0) 0 (0) 0 (0) 0 (0)
308 (89) 33 (83) 10 (100) 6 (100)
Number of all clonal cases (%) 237 (90) 165 (83) 156 (83) 36 (95) 27 (100) 20 (90) 11 (85) 9 (82) 5 (100) 2 (100)
MZL, marginal zone lymphoma; FL, follicular lymphoma; DLBCL, diffuse large B cell lymphoma; LPL, lymphoplasmocytic lymphoma; MCL, mantle cell lymphoma; B-CLL, lymphocytic lymphoma/leukemia; PLC, plasmacytoma; PTLD, posttransplant lymphoproliferative disorder; BL, Burkitt lymphoma; PMBCL, primary mediastinal B cell lymphoma; cHL, classical Hodgkin lymphoma; NLPHL, nodular lymphocyte predominant Hodgkin lymphoma; AITL, angioimmunoblastic T cell lymphoma; PTCL NOS, peripheral T cell lymphoma not otherwise specified; ALCL, anaplastic large cell lymphoma; MF, mycosis fungoides; NKTL, NK/T cell lymphoma; EATL, enteropathy associated T cell lymphoma; LA unspecified, unspecified lymphadenitis; Cutaneous PL, cutaneous pseudolymphoma; LyP, lymphomatoid Papulosis; Castleman’s, Castleman’s disease; Kikuchi, Kikuchi Lymphadenitis.
ordered by the diagnostic pathologist at the initial review of HE and Giemsa slides in all cases that did not show a classic morphological pattern. Final diagnoses were based on morphological and immunohistochemical findings in concert with results of clonality testing.
DNA extraction, multiplex PCR amplification and PCR product analysis Genomic DNA was extracted from 60 to 120 m of paraffinembedded tissue after deparaffinization using the QiaAmp Mini Kit (Qiagen, Hilden, Germany). Samples were divided into three groups based on DNA concentrations: <10 ng/l, 10–50 ng/l and >50 ng/l. The BIOMED-2 multiplex PCRs for the immunoglobulin heavy chain (IGH) VH –JH rearrangements (tubes A–C, IdentiClone, Invivoscribe Technologies, La Ciotat, France), as well as the multiplex reaction for T cell receptor gamma (TCRG) rearrangements (tubes A and B, IdentiClone), were performed as described previously [3] with 45 cycles. All samples were run in duplicates. Positive (clonal) and negative (polyclonal) controls in addition to a blank control (H2 O) were included in all cases. Gene scanning was carried out on an ABI-Prism 3100 Sequencer (Life Technologies, Darmstadt, Germany) using the Gene Scan Analysis Software 3.7. Cases were tested for IGH and/or TCRG rearrangements. The results were scored “clonal” if either the IGH or TCRG multiplex PCRs yielded a reproducible clonal product at the same position in both replicates. If apparent clonal peaks were of different fragment lengths in the two replicates (pseudoclonality), the analysis was scored not evaluable. The analysis was scored “clonal with polyclonal background” if the reactions showed a reproducible clonal product at least 3 times the intensity of the third largest peak observed.
Results Cases and diagnoses Of the 1969 cases tested for clonality, 1862 cases were tested for IGH and 1527 for TCRG clonality. Of these, 851 cases (43%) represented B cell lymphomas, 296 cases (15%) were diagnosed as T cell neoplasias, 89 cases were Hodgkin lymphomas (5%), 575 cases (29%) were finally classified as reactive lesions, and in 171 cases (9%), a non-lymphoid neoplasm was diagnosed or no definite diagnosis could be established. Detailed information about cases and diagnoses is found in Tables 1 and 2. Most specimens represented lymph nodes (511 cases). While B cell lymphomas dominated in all localizations except the skin (61–92%), T cell neoplasias (59%) were more frequently observed in skin than B cell lymphomas (41%). Interpretability of clonality analyses To assess the influence of the DNA concentration, we performed clonality analyses for Frameworks II and III of the IGH locus with cell line DNA (Raji cell line, a Burkitt lymphoma cell line) in different dilutions (starting from 446 ng/dl, 1:1, 1:3, 1:9, 1:27, 1:81, 1:243 and 1:729). We chose Frameworks II and III, since they are more frequently evaluable in primary samples than Framework I due to a shorter length of the PCR products. Optimal results for both Framework PCRs were obtained if the DNA concentration was ≥50 ng/dl (1:9 dilution), but reproducible peaks of the expected length could still be detected in both Framework reactions at a dilution of 1:81 (<10 ng/dl). To determine the influence of the DNA concentration on the interpretability of the clonality results, the samples were grouped according to DNA concentration: <10 ng/l, 10–50 ng/l and >50 ng/l in order to determine the influence of the DNA
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Table 2 TCRG rearrangements in the evaluable cases in different entities. Number of clonal cases without any polyclonal background (%)
Entity
Number of evaluable cases
B cell lymphomas (n = 448)
MZL FL DLBCL LPL MCL B-CLL PLC PTLD
124 110 147 26 15 15 5 6
Hodgkin (n = 70)
cHL NLPHL
58 12
1 (2) 1 (8)
T cell neoplasias (n = 266)
AITL PTCL NOS ALCL MF NKTL EATL SPLTL LyP
66 82 41 35 9 5 5 23
46 (70) 63 (77) 34 (83) 18 (51) 1 (11) 4 (80) 2 (40) 12 (52)
Reactive (n = 339)
LA unspecified Cutaneous PL Castleman’s Parapsoriasis Kikuchi
287 36 6 5 5
9 (3) 2 (6) 0 (0) 2 (40) 0 (0)
3 (3) 4 (4) 15 (10) 2 (8) 0 (0) 0 (0) 0 (0) 0 (0)
Number of clonal cases with polyclonal background (%)
Number of all clonal cases (%)
Number of oligoclonal cases (%)
Number of polyclonal cases (%)
31 (24) 26 (24) 38 (26) 4 (16) 4 (27) 3 (20) 3 (60) 1 (17)
34 (27) 30 (27) 53 (36) 6 (23) 4 (27) 3 (20) 3 (60) 1 (17)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
90 (73) 80 (73) 94 (64) 20 (76) 11 (73) 12 (80) 2 (40) 5 (83)
9 (16) 2 (17)
10 (17) 3 (25)
1 (2) 0 (0)
47 (81) 9 (75)
15 (23) 9 (11) 4 (10) 15 (43) 3 (33) 1 (20) 2 (40) 9 (39)
61 (92) 72 (88) 38 (93) 33 (94) 4 (44) 5 (100) 4 (80) 21 (91)
0 (0) 0 (0) 0 (0) 1 (3) 0 (0) 0 (0) 0 (0) 0 (0)
5 (8) 10 (12) 3 (7) 1 (3) 5 (56) 0 (0) 1 (20) 2 (9)
69 (24) 7 (19) 0 (0) 1 (20) 0 (0)
78 (27) 9 (25) 0 (0) 3 (60) 0 (0)
3 (1) 0 (0) 0 (0) 0 (0) 0 (0)
206 (72) 27 (75) 6 (100) 2 (40) 5 (100)
MZL, marginal zone lymphoma; FL, follicular lymphoma; DLBCL, diffuse large B cell lymphoma; LPL, lymphoplasmocytic lymphoma; MCL, mantle cell lymphoma; B-CLL, lymphocytic lymphoma/leukemia; PLC, plasmacytoma; PTLD, posttransplant lymphoproliferative disorder; BL, Burkitt lymphoma; cHL, classical Hodgkin lymphoma; NLPHL, nodular lymphocyte predominant Hodgkin lymphoma; AITL, angioimmunoblastic T cell lymphoma; PTCL NOS, peripheral T cell lymphoma not otherwise specified; ALCL, anaplastic large cell lymphoma; MF, mycosis fungoides; NKTL, NK/T cell lymphoma; EATL, enteropathy associated T cell lymphoma; SPLTL, subcutaneous panniculitis-like T cell lymphoma; LA unspecified, unspecified lymphadenitis; Cutaneous PL, cutaneous pseudolymphoma; LyP, lymphomatoid Papulosis; Castleman’s, Castleman’s disease; Kikuchi, Kikuchi Lymphadenitis.
yield non-interpretable results (7 and 5 samples for IGH and TCRG, respectively) compared to samples with >500 cells/section (2 and 3 samples for IGH and TCRG, respectively, Suppl. Tables 1 and 2). Comparably, the number of samples with monoclonal results was higher if the content of lymphoid cells/section was above 500 (16 and 9 samples for IGH and TCRG, respectively) compared to samples with <100 cells/section (2 and 1 sample(s) for IGH and TCRG, respectively). However, eleven B cell lymphoma samples with a content of <500 clonal cells/section still yielded reproducible clonal peaks in at least one Framework reaction.
Clonality in B cell lymphomas Fig. 1. Interpretability of clonality results in different assays. Samples are sorted by DNA concentrations (<10 ng/l, 10–50 ng/l, >50 ng/l). Percentages of evaluable clonality assays in relation to all cases tested are displayed. Frameworks I–III (tubes A–C) of the IGH VH –JH rearrangements (n = 1862), TCRG T cell receptor gamma (n = 1527). **p < 0.01, Chi-Square Test.
concentration on the interpretability of the clonality results. The interpretability of the results for the three regions Framework I–III of IGH VH –JH , as well as TCRG, was significantly dependent on the DNA concentrations (Fig. 1, Chi-Square Test p < 0.01). Therefore, in all the reactions, samples were more likely to be evaluable when the DNA concentration was above a critical threshold of 50 ng/l. Nevertheless, overall 64% of IGH and 56% of TCRG cases with a DNA concentration <10 ng/l still yielded evaluable results. Since the interpretability of the clonality assays does not only depend on the DNA concentration but also on the number of lymphocytes in the tissue, we assessed the number of B and T cells in the histologic section for all samples with a DNA content <10 ng/dl. Samples with a content of <100 cells/section of the respective lineage had a higher probability to
Mature B cell lymphomas showed clonal IGH VH –JH rearrangements in at least 83% of the evaluable cases. The highest frequencies of clonal IGH rearrangement rates (100%) were found in mantle cell lymphoma, mediastinal B cell lymphoma, and Burkitt lymphoma. Clonal IGH rearrangements were found in 83% of typical germinal center-derived follicular lymphoma (FL) and germinal center or post-germinal center-derived diffuse large B cell lymphoma (DLBCL). Monoclonal IGH rearrangements were seen in 85–95% of other “low grade” B cell lymphomas, including lymphocytic lymphoma (B-CLL), marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma, and plasmacytoma, which are in most cases post-germinal center-derived and have predominantly undergone the somatic hypermutation process (Table 1). Clonal rearrangements of the TCRG locus were most frequently found in DLBCL (36% of 147 evaluable DLBCL). All other B cell lymphoma entities (groups consisting of at least 15 cases) showed clonal rearrangements of the TCRG in ≤27% of the cases. All DLBCL with clonal TCRG rearrangements were retrospectively reviewed. At close inspection, among the 15 cases with a monoclonal TCRG rearrangement pattern without polyclonal background, seven cases were identified
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Fig. 2. Pleomorphic monoclonal T cell proliferation in a diffuse large B cell lymphoma, suggesting an underlying peripheral T cell lymphoma. (a) HE, 200×. (b) Sheets of B cell blasts. CD20-immunostaining, 200× (CD20, 1:1000, DAKO, Glostrup, Denmark). (c) CD3-immunostaining highlighting large pleomorphic T cells (arrows), 400× (CD3, 1:200, Novocastra, Newcastle upon Tyne, UK). (d) Monoclonal rearrangement of TCRG.
with either remarkably pleomorphic T cells or and aberrant T cell immunophenotype (Figs. 2 and 3), suggesting a true monoclonal T cell proliferation. Only two cases presented an epithelioid cell-rich background, consistent with an underlying angioimmunoblastic T cell lymphoma (AITL). Age distribution did not differ between DLBCL patients with or without clonal TCRG rearrangement (mean 62 vs. 61 years, respectively). Among the cases exhibiting a monoclonal TCRG peak with additional polyclonal background, no further evidence of a T cell neoplasia was found. Clonality in T cell neoplasias The various T cell lymphoma entities exhibited clonal rearrangements of the TCRG in the majority of cases (Table 2). Interestingly, AITL and mycosis fungoides (MF) showed the highest percentage of TCRG rearrangement (94% each), whereas only 88% of peripheral T cell lymphomas not otherwise specified (PTCL NOS) showed clonal rearrangements. Clonal VH –JH rearrangements of the IGH locus were rarely found in PTCL NOS and MF (16% and 20%, respectively). However, 29% of anaplastic large cell lymphomas and 25% of AITL showed clonal IGH rearrangements. Clonality in Hodgkin lymphoma Cases of classical and nodular lymphocyte predominant HL (NLPHL) exhibited similar frequencies of clonal rearrangements of the IGH locus (32% and 29%, respectively) and TCRG locus (17%
and 25%, respectively). All HL with clonal rearrangements of either IGH or TCRG were retrospectively reviewed. In cases of classical HL cases with a clonal IGH rearrangement, high numbers of Hodgkinand Reed–Sternberg cells were usually observed. In NLPHL clonal IGH rearrangements could not be explained by high numbers of tumor cells; however, in these cases, the clonal peak was always associated with a polyclonal background. Monoclonal TCRG rearrangements without a polyclonal background were only rarely observed in classical HL and NLPHL (one case each). Three cases originally classified as classical HL (with EBV-infected Hodgkin- and Reed–Sternberg cells) and two NLPHL with monoclonal TCRG rearrangement were reclassified as PTCL NOS or AILT after the review and were included in those categories for the remainder of the study.
Clonality in reactive lesions In a series of 598 reactive lesions, 459 were tested for rearrangements of the IGH locus and 362 for TCRG rearrangements. The vast majority of reactive lesions showed polyclonal patterns for the IGH locus (368 of 415 evaluable cases, 89%), as well as for the TCRG (248 of 362 evaluable cases, 69%). Monoclonal rearrangements of the IGH locus occurred in 36 cases (10%); of these, there were 9 cases in which a monoclonal product without polyclonal background was detected in at least one framework (2% of all reactive lesions). Three of these cases were lymph nodes from children showing massive follicular hyperplasia. Clinical follow up data (1
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Fig. 3. Monoclonal T cell proliferation with an aberrant immunophenotype in a diffuse large B cell lymphoma, suggesting an underlying peripheral T cell lymphoma. (a) HE, 200×. (b) Nodular accumulation of B cell blasts in CD20 immunostaining, 100×. (c) CD3-positive T cells surrounding the nodules of B blasts, 100×. (d) CD10-immunostaining highlighting the T cells, while B cell blasts remain negative, 100× (CD10, 1:100, Novocastra). (e) Ki67-immunostaining showing an elevated proliferation rate, particularly in the B cell blasts, 100× (Ki67, 1:200, DAKO). (f) Monoclonal rearrangement of TCRG.
month–4 years) were available for four additional patients. None of these patients developed a lymphoma. However, since pediatric follicular lymphoma often presents with a localized stage, can be BCL2-negative [6], and can be cured by simple lymphadenectomy, we cannot completely rule out the infiltration by pediatric follicular lymphoma in the three children. The majority of reactive lesions with clonal rearrangement patterns (27/36 cases, 75%) presented a polyclonal background, and in 20 of these cases (56%), the clonal amplificate was restricted to one of the three VH rearrangement PCRs (Fig. 4). In contrast, B cell lymphomas more frequently exhibited a clear monoclonal rearrangement in at least two VH PCR assays without a concomitant polyclonal background (444 cases, 66%,
p < 0.05, Fishers Exact Test, Fig. 4). Compared to polyclonal reactive lesions, reactive lesions with clonal IGH rearrangements were more likely, albeit not significantly, to be localized in the gastrointestinal tract (11% vs. 4%) or respiratory tract (36% vs. 20%). Clonal rearrangements of the TCRG in reactive lesions were observed approximately at the same frequency as clonal rearrangements of the IGH locus (90 of 339 cases, 27%). Clear monoclonal products without any polyclonal background were found in 9 nonspecific reactive lesions (3% of all reactive lesions). Two of these patients were HIV-positive, and one was hepatitis C-positive. Clinical follow up data (5 months–7 years) were available in three patients. None of the patients developed a lymphoma. Interestingly, the
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Fig. 4. Pattern distribution of clonally rearranged IGH framework regions in B cell lymphomas and reactive lesions. (a) Typical monoclonal product without polyclonal background in Framework PCR II (tube B of IGH) in a B cell lymphoma. (b) Typical pattern of monoclonal product with polyclonal background in Framework PCR II (tube B of IGH) in a non-specific lymphadenitis. (c) B cell lymphomas and reactive lesions with clonally rearranged IGH loci, sorted by the number of clonally rearranged framework PCRs and the presence of additional polyclonal background. **p < 0.01, *p < 0.05, Fisher’s Exact Test.
frequency of clonal rearrangements of the IGH locus or the TCRG locus in cutaneous pseudolymphoma (18% and 25%, respectively) was similar to the frequencies of clonal IGH and TCRG rearrangements observed in nonspecific reactive lesions of other localizations (15% and 27%, respectively). Discussion Clonality testing is performed in many laboratories using the BIOMED-2 primers [3], and results are highly comparable between laboratories. We evaluated a large cohort of consultant cases and primary diagnostic samples tested for clonality. Selection criteria for clonality testing were either to confirm the conventional diagnosis or to resolve differential diagnostic questions. The data may be biased, since the final diagnosis included the results of clonality testing; however, the overall results match very well with the findings observed by others: in the present study, clonal IGH VH –H rearrangements were observed in 83% of each the FL and DLBCL in the evaluable cases; in the study by Evans et al. [1], 84% and 79% of the FL and DLBCL, respectively, showed clonal IGH VH –H rearrangements. Other investigators observed lower percentages of IGH rearrangements: in FL for example, frequencies ranged from 31% to 80% when only the IGH approach was applied [7–10]. These differences may be due to different technical reasons. We are also aware that because of somatic hypermutation of the IGH locus [3], the sensitivity of clonality detection can be dramatically increased by the light chain assays [1,7,8,10].
In line with previous investigations [10], we found the DNA concentration to be an important factor for the interpretability of the data. Nonetheless, still 53% of samples with DNA contents <10 ng/l showed evaluable results for VH -FR3–JH . Pseudoclonal results, likely to be obtained in samples containing only small numbers of lymphocytes [11], were scored not evaluable. A content of >500 lymphoid cells per section of the respective lineage proved to be critical for reliable PCR results. However, in rare cases of B cell lymphomas, clonal patterns for IGH could be demonstrated even if the content of clonal cells per section was <500. This indicates that the number of cells required for an interpretable result is less in a monoclonal than in a polyclonal population. Therefore, a clonality test always seems to be worth trying. However, due to the low number of samples with a DNA content below 10 ng/dl, these results should be interpreted with caution. Interestingly, an additional clonal TCRG rearrangement was most frequently detected in DLBCL (36%, Table 2). In the study by Evans et al. [1], clonal rearrangements of the TCRG were likewise most frequently observed in DLBCL, extranodal MALT lymphoma, and B-CLL (15–18%). In the present study, seven of these cases showed a T cell infiltrate either with pleomorphism or with an aberrant immunophenotype, suggesting an underlying T cell lymphoma which was overgrown and effaced by an emerging large B cell lymphoma. In all other cases with clonal TCRG rearrangements, no morphologic correlate of the T cell clone could be identified. Therefore, DLBCL with clonal TCRG rearrangements can be divided into two groups: on the one hand DLBCL arising in
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previously existing T cell lymphoma, particularly AITL or PTCL NOS [12–14]. On the other hand DLBCL developing on a background of an impaired immune control for other reasons, as can be observed in the elderly or in immunocompromised individuals. Dojcinov et al. [15] observed restricted or monoclonal T cell repertoires in 30% of EBV-positive DLBCL. A similar issue was addressed in a study by Ibrahim et al. [16], who found clonal T cell expansions in 50% of PTLD. Hence, the number of DLBCL arising in an immunocompromised background with impaired T cell control of the malignant clone may be higher than expected. Another explanation for clonal TCRG rearrangements in DLBCL may be a very low T cell content in the tissue of some cases. We observed similar rates of clonal TCRG rearrangements in PTCL NOS, ALCL, and AITL as noted in previous reports [17,18]. For AITL, this is surprising, as previous studies [19] showed that the neoplastic population may be a minority in AITL. B cell clonality (IGH) in T cell lymphomas was detected in the present study in 19–29% (Table 1), which is in line with previously reported rates of 30% in PTCL NOS [20], 9–11% in ALCL [18], and 19% in AITL [17]. Emerging B cell lymphomas have been described in many T cell lymphoma subsets [12,13,18,21], and monoclonal IGH rearrangements may be indicative of an early B cell expansion due to the impaired control of the immune system in the setting of a T cell neoplasia. This may particularly be the case for EBV-infected B cell clones. We detected clonality for IGH in approximately 1/3 of the HL cases. In 19% of classical HL, clonal products lacking polyclonal background were found, which could morphologically be explained by an abundant tumor cell infiltrate. IGH clonality in classical HL was previously reported in 16% [22], 21% [23], 40% [24], and 67% [25] of the cases. In all NLPHL showing clonal IGH rearrangements, the clonal product was found to be associated with a polyclonal background infiltrate, reflecting an abundance of reactive B cells in the infiltrate. Interestingly, of four original classical HL with a monoclonal TCRG product, after histopathologic review, three cases had to be reclassified as T cell lymphomas (2 as PTCL NOS with Hodgkin- and Reed–Sternberg cells and 1 AITL), and two NLPHL were reclassified as PTCL NOS. There are contradictory data regarding clonality of rosetting T cells around LP cells [26,27]. However, the number of cases investigated by microdissection of rosetting T cells is very low. Previously described atypical T cells occurring in NLPHL were reported to be polyclonal [28]. Therefore, HL with a clear monoclonal TCR rearrangement very likely represents particular PTCL with a “Hodgkin-like” morphologic presentation [29]. Among reactive lesions, clonal TCRG rearrangements with a polyclonal background infiltrate were quite frequent (23%). This phenomenon is very likely due to an antigen-driven clonal selection. Clonal rearrangements lacking a polyclonal background were rarely found among reactive lesions (3%) compared to nodal T cell lymphomas (where >70% lacked a polyclonal background). For the IGH locus, clonal products were even rarer; only 2% of all reactive lesions showed a clonal product for IGH without a polyclonal background infiltrate. Similar results were obtained by Langerak et al. who detected a clear monoclonal product for one Immunoglobulin or TCR target in 10% of reactive samples [2]. In our analysis, reactive lesions with clonal IGH patterns in most cases included an additional admixture of polyclonal products (8%), and clonality was mostly observed only in one or two Framework PCRs only. This is likely due to a preferential PCR amplification of one clone within a polyclonal infiltrate, which is more likely to happen in only one or two Framework PCRs than in all three reactions. Additionally, the localization of the diagnostic biopsy should be taken into account. We more often observed clonal IGH amplificates in reactive lesions from extranodal localizations, like gastrointestinal or respiratory tract. One reason may be the extensive exposure of mucosal
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surfaces to antigens that might stimulate the proliferation of specific B cell clones [30,31]. In summary, the present study shows that the BIOMED-2 primers represent a powerful diagnostic tool for assessing the clonality of B and T cells in reactive and neoplastic conditions. By careful examination of the rearrangement patterns, including an assessment of a co-amplified polyclonal background, an assessment of clonal rearrangements among multiple PCR reactions, as well as considering the localization of the biopsy, important information can be obtained, which in concert with morphologic and immunohistochemical parameters can lead to a firm diagnosis. Conflict of interest The authors report no potential conflict of interest. Acknowledgements The authors would like to thank Susanne Hansen for her excellent technical assistance. The authors thank Prof. Ralf Küppers, Essen, Germany, for his helpful discussions and comments on the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prp.2013.05.004. References [1] P.A. Evans, C. Pott, P.J. Groenen, G. Salles, F. Davi, F. Berger, et al., Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936, Leukemia 21 (2007) 207–214. [2] A.W. Langerak, T.J. Molina, F.L. Lavender, D. Pearson, T. Flohr, C. Sambade, et al., Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936, Leukemia 21 (2007) 222–229. [3] J.J. van Dongen, A.W. Langerak, M. Bruggemann, P.A. Evans, M. Hummel, F.L. Lavender, et al., Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936, Leukemia 17 (2003) 2257–2317. [4] R.F. McClure, P. Kaur, E. Pagel, P.D. Ouillette, C.E. Holtegaard, C.L. Treptow, et al., Validation of immunoglobulin gene rearrangement detection by PCR using commercially available BIOMED-2 primers, Leukemia 20 (2006) 176–179. [5] H. Liu, A.J. Bench, C.M. Bacon, K. Payne, Y. Huang, M.A. Scott, et al., A practical strategy for the routine use of BIOMED-2 PCR assays for detection of B- and T-cell clonality in diagnostic haematopathology, Br. J. Haematol. 138 (2007) 31–43. [6] I. Oschlies, I. Salaverria, F. Mahn, A. Meinhardt, M. Zimmermann, W. Woessmann, et al., Pediatric follicular lymphoma – a clinico-pathological study of a population-based series of patients treated within the Non-Hodgkin’s Lymphoma–Berlin–Frankfurt–Munster (NHL-BFM) multicenter trials, Haematologica 95 (2010) 253–259. [7] A.M. Halldorsdottir, B.A. Zehnbauer, W.R. Burack, Application of BIOMED2 clonality assays to formalin-fixed paraffin embedded follicular lymphoma specimens: superior performance of the IGK assays compared to IGH for suboptimal specimens, Leuk. Lymphoma 48 (2007) 1338–1343. [8] K. Payne, P. Wright, J.W. Grant, Y. Huang, R. Hamoudi, C.M. Bacon, et al., BIOMED-2 PCR assays for IGK gene rearrangements are essential for B-cell clonality analysis in follicular lymphoma, Br. J. Haematol. 155 (2011) 84–92. [9] Y.L. Chen, I.J. Su, H.Y. Cheng, K.C. Chang, C.C. Lu, N.H. Chow, et al., BIOMED-2 protocols to detect clonal immunoglobulin and T-cell receptor gene rearrangements in B- and T-cell lymphomas in southern Taiwan, Leuk. Lymphoma 51 (2010) 650–655. [10] E. Berget, L. Helgeland, A. Molven, O.K. Vintermyr, Detection of clonality in follicular lymphoma using formalin-fixed, paraffin-embedded tissue samples and BIOMED-2 immunoglobulin primers, J. Clin. Pathol. 64 (2011) 37–41. [11] A. Boer, R. Tirumalae, M. Bresch, T.M. Falk, Pseudoclonality in cutaneous pseudolymphomas: a pitfall in interpretation of rearrangement studies, Br. J. Dermatol. 159 (2008) 394–402. [12] K. Willenbrock, A. Brauninger, M.L. Hansmann, Frequent occurrence of Bcell lymphomas in angioimmunoblastic T-cell lymphoma and proliferation of Epstein–Barr virus-infected cells in early cases, Br. J. Haematol. 138 (2007) 733–739.
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