Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell receptor (TCR) rearrangements can be useful. Here we address the issue of clonality assessment in 106 histologically defined reactive lesions, using the standardized BIOMED-2 Ig/TCR multiplex polymerase chain reaction (PCR) heteroduplex and GeneScan assays. Samples were reviewed nationally, except 10% random cases and cases with clonal results selected for additional international panel review. In total 75% (79/106) only showed polyclonal Ig/TCR targets (type I), whereas another 15% (16/106) represent probably polyclonal cases, with weak Ig/TCR (oligo)clonality in an otherwise polyclonal background (type II). Interestingly, in 10% (11/106) clear monoclonal Ig/TCR products were observed (types III/IV), which prompted further pathological review. Clonal cases included two missed lymphomas in national review and nine cases that could be explained as diagnostically difficult cases or probable lymphomas upon additional review. Our data show that the BIOMED-2 Ig/TCR multiplex PCR assays are very helpful in confirming the polyclonal character in the vast majority of reactive lesions. However, clonality detection in a minority should lead to detailed pathological review, including close interaction between pathologist and molecular biologist.
Lymphoproliferative disorders are generally diagnosed based on histomorphological and immunophenotypical criteria. Although data obtained with these methods are mostly conclusive, in 5–15% of cases the differential diagnosis between reactive lymphoproliferative lesions and malignant lymphomas is difficult, requiring complementary methods. In such cases molecular clonality studies of immunoglobulin (Ig) and/or T-cell receptor (TCR) gene rearrangements can be a useful additional tool. Ig/TCR gene rearrangements occur sequentially in the earliest stages of lymphoid differentiation and thus are present in almost all immature and mature lymphoid cells. As lymphomas and leukemias are derived from a single malignantly transformed lymphoid cell, virtually all of them contain one or several clonal Ig and/or TCR gene rearrangements.
In a recent European collaborative study (BIOMED-2 Concerted Action BMH4-CT98-3936) new standardized multiplex polymerase chain reaction (PCR) strategies for more reliable analysis of Ig/TCR gene rearrangements were designed.1 In addition, multiplex assays for detection of the well-defined t(11;14) and t(14;18) chromosome aberrations were developed, which can also be helpful for clonality assessment. Following initial validation of these standardized multiplex assays on a limited set of Southern blot (SB) defined samples,1 the patterns of (in)complete Ig/TCR gene rearrangements and t(11;14) and t(14;18) aberrations have now been assessed in full detail in different types of B- and T-cell lymphomas as defined according to the World Health Organization (WHO) classification.2, 3, 4
Although such information will help to define the value of analyzing individual PCR targets for clonality assessment in different lymphoma entities, another major issue concerns the meaning of data obtained with these newly developed multiplex PCR assays in histomorphologically defined reactive tissue lesions. In biological terms, histologically reactive lesions represent a broad spectrum of lesions ranging from heterogeneous, polyclonal lymphocytes, via true reactive lymphoproliferations, to proliferations containing (oligo)clonally activated lymphoid cell populations or even a monoclonal component.5, 6, 7, 8, 9, 10, 11 Reactive lymphoproliferations in lymph nodes or other tissues arising upon infection with micro-organisms (e.g. Epstein–Barr virus (EBV), cytomegalovirus, human immunodeficiency virus infections, toxoplasmosis, tuberculosis, cat scratch disease) or being associated with particular inflammatory processes (e.g. sarcoidosis, Hashimoto's thyroiditis, myoepithelial sialo-adenitis) are thought to result from activation of polyclonal antigen-selected lymphocytes.12, 13, 14, 15, 16, 17, 18, 19, 20 It is largely unknown how such (massive) antigen-driven proliferations influence Ig/TCR clonality assessment. In particular the question arises whether all histologically defined reactive lesions are truly polyclonal lymphoproliferations or whether oligoclonal or even seemingly monoclonal gene rearrangement patterns might be found, owing to the high pressure of chronic antigen-driven selection of B cells. On the other hand, clonal Ig/TCR rearrangements might also represent truly clonal cell populations in a background of polyclonal reactive cells.10 The latter may have impact on early diagnosis and/or assessment of lymphoma/leukemia spreading to other tissue locations.
Here we address the issue of Ig/TCR clonality in histomorphologically reactive lesions using the standardized BIOMED-2 multiplex PCR clonality assays and discuss its meaning in diagnosing reactive lesions. To this end, initially a total of 109 fresh-frozen reactive tissue samples from various national networks of the BIOMED-2 Concerted Action were included for extensive PCR analysis of all possible Ig/TCR gene rearrangements as well as t(11;14) and t(14;18) aberrations. Samples were reviewed nationally, except cases with unexpected molecular results, which were selected for additional panel review. The results show that ∼75% of all cases are clearly polyclonal for all Ig/TCR targets. In another 15% of cases some doubtful molecular results were observed for one or two targets, which could mostly be explained technically; so these samples are most probably polyclonal as well. However, in 10% of the samples clear monoclonal products were observed for one or more Ig/TCR targets, which prompted further pathological review; these cases are presented and discussed in detail here. Finally, the diagnostic implications of finding Ig/TCR clonality in histomorphologically reactive tissue samples are evaluated.
Materials and methods
Inclusion of samples and review process
Initially, a total of 109 reactive tissue lesions were included in this study (Table 1). In the study design we excluded paraffin-embedded samples, as the PCR results of such samples are largely dependent on variation in formalin fixation and paraffin embedding, which was beyond control of this study. Most of these 109 samples concerned consecutively collected biopsies that were taken because of initial clinical suspicion of malignant lymphoma, and which were considered reactive in histomorphology and immunohistochemistry. Approximately 37 reactive tissue lesions were collected retrospectively. Samples were only included if sufficient frozen tissue was available for the molecular assays. The vast majority of samples concerned lymph node biopsies (n=90; six axillary, 13 cervical, 20 occipital, five submandibular, seven inguinal, one mediastinal, one mesenteric, one abdominal, one para aortic and 35 of unspecified sites), whereas other biopsies were from appendix (n=1), sinus maxillaris (n=1), skin (n=4), spleen (n=5), thyroid gland (n=1) and tonsil (n=7). Owing to clerical errors four samples (one axillary lymph node, one cervical lymph node, one inguinal lymph node and one tonsil) had to be excluded from the reactive lesion series before PCR analysis, whereas for the same reason one lymph node sample was added that was initially included in one of the lymphoma series.3, 4
Initial diagnosis of the samples was made by the local pathologist, followed by reviewing at the national level by a panel of pathologists for virtually all samples. According to the study guidelines approximately 10% of the samples were randomly selected for international review by the BIOMED-2 Pathology Review Panel. Finally, following molecular analysis, cases with unexpected results in the molecular assays were selected by the network leader (AWL) and the coordinating pathologist (JHJMvK) for further review. In this way, 17 additional samples were internationally reviewed at a later stage in the study by the BIOMED-2 Pathology Review Panel.
Multiplex PCR amplification and PCR product analysis
Following the predefined inclusion criteria 106 clinically suspect but histomorphologically reactive tissue biopsies were included in the study. Genomic DNA was isolated from these frozen tissue biopsies and DNA samples were distributed in series of approximately 30 cases to two different laboratories. In each laboratory all DNA samples of a series were studied in duplicate for signs of clonality using the complete panel of BIOMED-2 multiplex PCR assays.1 The BIOMED-2 multiplex tubes are available from InVivoScribe Technologies, San Diego, CA (www.invivoscribe.com).
The BIOMED-2 multiplex PCR assays included IGH (n=3+2 tubes; for V–J and D–J gene rearrangements, respectively), IGK (n=2), IGL (n=1), TCRB (n=2+1; for V–J and D–J gene rearrangements, respectively), TCRG (n=2) and TCRD (n=1) gene rearrangements and t(11;14) (n=1) and t(14;18) (n=3) chromosome aberrations and were performed according to standardized protocols and primers, as recently described.1 Following amplification, the Ig/TCR PCR products were evaluated by heteroduplex analysis or GeneScan analysis, as described previously.1, 21, 22 Every sample was analyzed by two paired laboratories participating in the BIOMED-2 Concerted Action (first set of samples: Cambridge and Toulouse; second set: Rotterdam and Paris; third set: Groningen and Porto; fourth set: Southampton and Heidelberg). Within each laboratory every sample was also analyzed in duplicate, that is, once via heteroduplex analysis and once via GeneScan analysis (Cambridge, Rotterdam, Groningen, Porto, Southampton) or twice via heteroduplex analysis (Toulouse, Paris, Heidelberg) for Ig/TCR rearrangements; for analyzing the two chromosome aberrations the duplicate PCR products were analyzed with agarose gel electrophoresis. In case of discrepant PCR results between the two laboratories, a third laboratory was involved to repeat the assay for that particular target.
To score samples as presenting with clonality, low-level clonality or polyclonality, some general guidelines were applied. Clonality was defined as unequivocal clonal Ig/TCR bands/peaks; in principal such clonal bands should be present in multiple multiplex PCR reactions, although occasionally clear clonality would be seen in a single multiplex Ig/TCR PCR. Low-level clonality was defined as the presence of an equivocal, mostly weak Ig/TCR band/peak within a background of polyclonal rearrangements. Moreover, such equivocal products would often be detected with either heteroduplex or GeneScan analysis only. Finally, clear polyclonality concerns polyclonal patterns in all multiplex PCR reactions.
For the 106 samples together a total of 106 (number of samples analyzed) × 14 (number of multiplex Ig/TCR PCR tubes) × 2 (number of laboratories evaluating a given sample) × 2 (all analyses in duplicate) ∼6000 PCR reactions were performed. Analysis of the t(11;14) and t(14;18) aberrations resulted in an additional 1696 (106 × 4 × 2 × 2) PCR reactions.
Intra-laboratory variation between two independent duplicate Ig/TCR PCR assays was limited and occurred in 34 of the roughly 3000 (=∼1%) PCR duplicates; these variations mostly concerned (weak) clonal products being detected in GeneScan analysis but not in heteroduplex analysis, although especially for TCRB Vβ–Jβ analysis the discordancy was often the other way round. Such intra-laboratory variation was often resolved when data were compared to those of the second laboratory; in occasional cases in which intra-laboratory variation could not be resolved this way, a third laboratory was involved to repeat the assay (see also later). As such variation between the two laboratories analyzing the same set of samples was also relatively limited, with inter-laboratory discrepancies being observed in 70 of the ∼3000 (=∼2%) duplicate PCR tests. In case of discordant inter-laboratory results, a third laboratory repeated the PCR assay in order to come to final conclusions on polyclonality or clonality. Based on the collective results of all PCR assays, the 106 cases were subsequently classified as: type I, clear polyclonal samples (polyclonal in all PCR reactions); type II, probably polyclonal samples (low-level clonality mainly in a background of polyclonal Ig/TCR rearrangements; this usually concerned a weak clonal band in a limited number of multiplex PCRs and mostly only detected with a single technique); type III, probably clonal samples (clear clonality in a single multiplex PCR and/or single Ig/TCR locus); type IV, clear clonal samples (clear clonality pattern in multiple multiplex PCRs).
The results obtained in the t(14;18) and t(11;14) analysis in the series of 106 reactive lesions were consistently negative in all samples.
Polyclonal and probably polyclonal tissue biopsies
The vast majority of histomorphologically reactive tissue samples (79/106, 75%) were categorized as type I samples. Only polyclonal Ig/TCR PCR products were found in all multiplex PCR reactions and also no t(11;14) or t(14;18) PCR products were detected, thus confirming the histomorphological conclusions. In 16 of 106 cases (15%) low-level Ig/TCR clonality was observed (Table 2), mainly concerning a minor population in a polyclonal background. Six cases (NL 148, NL 101, NL 132, NL 262, FR 173 and ES 196) showed clonality in a single IGH VH–JH or IGH DH–JH PCR only, observed in only one method of evaluation (heteroduplex or GeneScan analysis). In some other cases (NL 132, NL 153, NL 157 and ES 191) a similar phenomenon was observed for TCR clonality analysis, with clonal TCRB or TCRG products being detected with heteroduplex or GeneScan analysis only; moreover, in the TCRB-positive cases no parallel TCRG clonality was observed and in the TCRG-positive cases no parallel TCRB clonality was found. Finally, six cases (NL 158, NL 259, ES 126, PT 004, PT 020 and GBS 095) were remarkable in the sense that only TCRD clonality was observed without any evidence for clonal TCRB or TCRG products. This is highly suggestive for pseudoclonality, caused by selective amplification of a very restricted TCRD repertoire or the presence of only few cells with TCRD rearrangements in these samples. Similarly, the results in case NL 264, which was found to be clonal in TCRG tube B only, should be explained by pseudoclonality as well, as this PCR tube predominantly detects the very rare TCRG rearrangements. As clonality detection in these 16 cases was concluded to be at least doubtful, these cases were labeled type II tissues, or probably polyclonal reactive tissues.
Histomorphologically reactive tissue samples showing Ig/TCR clonality
In contrast to the type II samples with doubtful clonality results, 10% (11/106) of all samples showed clear monoclonal products for one Ig/TCR target (Table 3). The samples NL 172 and GBN 037 only showed clear clonal IGK PCR products, but without any signs of clonality upon IGH VH–JH and DH–JH analysis. One other sample (NL 156) did show clonality in three different TCR loci, but the TCRB Dβ–Jβ rearrangements were seen in GeneScan analysis only. Although the latter might reflect true clonality, pseudoclonality owing to low levels of template for PCR amplification cannot be completely excluded, also given the weak TCRD products seen in this sample. Nevertheless, these three cases were all suspect of containing a true clone and were categorized as probably clonal (type III) tissue samples.
The remaining eight samples were type IV samples showing clear clonality in multiple Ig (NL 108, PT 021 (Figure 1 a–c), GBN 017 and DE 105) or TCR (NL 111 (Figure 2 a–c), PT 024, DE 069 and DE 107) loci, indicative of clear B- and T-cell clones, respectively. In two samples (PT 021, DE 105) additional clonal TCRB products were seen on top of the Ig clonality, but these were mostly weak and in one case only observed with a single technique. This phenomenon is regularly seen in lymphoma samples (Evans et al, this issue)3 and therefore did not influence the interpretation of the presence of the clonal B-cell population.
Additional SB analysis was performed on the type III/IV samples to confirm their clonal character as determined by PCR. Indeed, in four out of 11 samples the SB results clearly confirmed the presence of a B- or T-cell clone clone (Table 3). In the remaining cases no clone could be identified by SB analysis, which is most probably related to the small clone size, being below the SB detection limit.2
Detailed histological characterization of samples with clonal Ig/TCR gene rearrangements
Cases with unexpected clonal results (type III and IV cases) were reviewed in the international pathology review panel (Table 3). In this review process two cases (DE 069, DE 105) were identified as representing a lymphoma with partial involvement of the lymph node. One case (DE 069) concerned an enlarged lymph node in a patient with mycosis fungoides, having a dermatopathic appearence but with large cerebriform cells being present. The other case (DE 105) was a lymph node with marginal zone lymphoma that had remaining large germinal centers. Both samples therefore represent known difficult situations to pathologists, for which clonality analysis was thus very useful as quality control for making the final diagnosis.
The other 10 type III/IV cases could not be resolved in a straightforward manner (Table 3). Firstly, frozen tissue sections were reviewed to exclude sampling problems. In one case (GBN 017) the frozen tissue sample did contain a large germinal center. As it is known that germinal centers often consist of B-lymphocytes from as few as one to five clones,23 this might explain the molecular clonality results. This case illustrates the importance of checking the constitution of the tissue from which the DNA is extracted by evaluating a frozen tissue section. Next, the enlarged lymph node (DE 107) from a patient known to have been treated for diffuse large B-cell lymphoma (DLBCL) at an earlier stage was reviewed and still regarded reactive, despite knowledge of the molecular results. Moreover, as the observed clonality concerned the TCR loci, this cannot be a localization of the DLBCL, and so the molecular findings remained unexplained.
The remaining cases concerned biopsies from patients without a diagnosis of lymphoma but with clear clonal Ig/TCR results. Some of these represent well-known pitfalls such as pseudolymphoma of the skin vs minimal localization of marginal zone skin lymphoma (NL 108, GBN 37), and idiopathic thrombocytopenic purpura (NL 156) which concerns reactive processes in which oligoclonal B-cell proliferations and sometimes one predominant clone can be found. As we did not see clear Ig light chain restriction in the skin pseudolymphomas, based on the prevailing histologic criteria these cases could not be diagnosed as marginal zone skin lymphomas. For such cases the interaction between the pathologist and the molecular biologist is crucial and all clinical data need to be taken into account. There also was one case of progressively transformed germinal centers (NL 172). It has been described that in such cases clonal populations can be found similar to normal germinal centers.24 Case PT 024 with clonal TCRB and TCRG rearrangements showed an enlarged lymph node with an atypical hyperplasia of the paracortex, having features of early angio-immunoblastic T-cell lymphoma (AILT) although being present only focally in the lymph node. Furthermore, CD21 staining did not show expanded dendritic networks. Although this case could thus not be diagnosed as lymphoma based on WHO histomorphology criteria only, the molecular results do point in that direction.
Finally, two cases of ruptured spleen remained, both showing an enlarged spleen but with a normal architecture and white pulp. Only with knowledge of the molecular results and after additional immunohistochemical staining an abnormal cell population could be found in both cases. Case NL 111 with clonal TCRB and TCRG gene rearrangements (Figure 1 a–c) an increase of granzyme-positive cytotoxic T-cells (Figure 1 d and e) was observed. In the other (PT 021), with IGH and IGL clonality (Figure 2 a–c), plasmacytosis with dominant cytoplasmic Igλ staining was seen (Figure 2 d–f). Unfortunately, we have no further clinical data of these patients, but the first case may concern T-cell large granular lymphocytosis, whereas the second may represent a form of plasma cell dyscrasia.
Several studies have documented the performance, reliability and value of the recently developed, standardized BIOMED-2 multiplex PCR assays for detecting (clonal) Ig/TCR gene rearrangements and t(11;14) and t(14;18) aberrations in well-defined leukemia and lymphoma cases.1, 2, 3, 4, 25 This information helps to define the value of using individual PCR targets for clonality assessment in the different lymphoma entities, but does not answer the question of clonality detection in histomorphologically defined reactive tissue lesions, which has only been addressed in a limited way so far.10, 26
Here we have addressed the issue how frequently Ig/TCR clonality can be observed in histomorphologically reactive lesions and what impact the clonality findings have with respect to the final diagnosis. To this end we have extensively analyzed 106 reactive tissue samples using the BIOMED-2 multiplex PCR assays for Ig/TCR gene rearrangements and t(11;14) and t(14;18) aberrations. The vast majority (75%; 79/106) of histomorphologically reactive samples indeed showed clear polyclonality for all Ig/TCR targets and negativity in t(11;14) and t(14;18) PCR. Hence, interpretation in these so-called type I cases was clear because they actually confirm the histomorphological diagnosis. In another 15% (16/106) of cases doubtful clonality results were observed for one or maximally two Ig/TCR targets (type II cases). However, these doubtful results could mostly be explained in a technical or immunobiological way: weak products, not completely polyclonal pattern in GeneScan or heteroduplex analysis, restricted TCR repertoire with the more rare TCRG and TCRD rearrangements becoming seemingly clonal. Therefore, these type II cases were considered as probably polyclonal. Such cases need careful evaluation of the histopathology in combination with the molecular Ig/TCR results. The type II cases with isolated TCRD clonality illustrate that TCRD analysis can lead to false-positive results. Hence, TCRD is not the reliable PCR target for evaluating T-cell clonality when considered without information from TCRG and preferably also TCRB locus evaluation. So, either TCRG or TCRB analysis, or preferably both, should accompany TCRD analysis. However, in practice this implies that the main application and contribution of TCRD analysis lies in analyzing clonality in suspected T-cell proliferations known to express a TCRγδ receptor.
Finally, clear monoclonal products were observed for one or more Ig/TCR targets in 10% (11/106) of the histomorphologically reactive samples. Depending on the number of Ig/TCR loci involved, these cases were considered probably clonal (type III; involvement of single locus) and clearly clonal (type IV; multiple loci involved). Even though we initially did not include SB analysis for all samples owing to its lower sensitivity as compared to PCR, additional SB analysis on the type III/IV cases showed a clear B- or T-cell clone in PCR in four samples (the two lymphomas, and the cases with progressively transformed germinal centers and one large germinal center). The fact that in the remaining cases no clone could be identified by SB analysis, is most probably related to their small clone size below the SB detection limit.2
Furthermore, all type III/IV cases with unexpected clonality were selected for additional international review. The BIOMED-2 Pathology Review Panel concluded partial lymph node involvement of lymphoma (1 × mycosis fungoides, 1 × MZL) in two of these 12 cases. Keeping in mind that the international review process was performed with full knowledge of the molecular results, one should conclude that Ig/TCR clonality would have been very useful in these two cases, as the lymphoma involvement had initially been missed in local and national review. Clonality findings in the other cases could often be explained as well-known pitfalls (clonality in e.g. skin pseudolymphoma, single large germinal center, transformed germinal center) or as probable lymphoma only upon pathology panel review. The latter was concluded in a case with atypical hyperplasia of the paracortex and features of early AILT. Upon international review no diagnosis of lymphoma was made based on histomorphology only, but the Ig/TCR clonality results point towards probable lymphoma involvement in this case. The same was true for the two cases of ruptured spleen (enlarged but with a normal architecture and white pulp) in which knowledge of the molecular results and subsequent additional immunohistochemical stainings identified abnormal cell populations (× 1 cytotoxic lymphocytes, × 1 Igλ-positive plasma cells).
Our data show that the BIOMED-2 Ig/TCR multiplex PCR assays are very helpful in confirming the polyclonal, reactive character in the vast majority of reactive lesions. Clonality detection in a minority of such cases should lead to close pathological review of the sample and close interaction between the pathologist and the molecular biologist to avoid potential pitfalls. Logically, in daily practice molecular Ig/TCR analysis is not required for every case. As a guideline it can be stated that clonality assessment is warranted for those samples that remain clinically suspect (despite reactive morphology) and/or remain histomorphogically unclear. It is estimated that this would apply for ∼5% (for experienced pathologists) to ∼15% (for less experienced pathologists) of the cases. If the results of PCR-based clonality detection is fully polyclonal, then the interpretation is straightforward. However, if the observed pattern is not completely polyclonal or clearly not polyclonal, first (technical and immunobiological) explanations should be evaluated between the pathologist and molecular biologist. In some cases the (oligo)clonality pattern can indeed be explained in technical terms (e.g. weak products, restricted repertoire) or by the underlying pathophysiology (e.g. progressively transformed germinal center, EBV infection). However, if the Ig/TCR clonality results remain suspicious, clinical follow-up with new tissue sampling and subsequent pathological and molecular analysis should be considered to reach a final diagnosis.
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We thank Ingrid LM Wolvers-Tettero, Ellen J van Gastel-Mol, Monique ECM Oud, Brenda Verhaaf (Rotterdam), Francis Devez (Paris), Helen White (Southampton), Jonathan Langdown, Karen Brown (Cambridge), Patrícia Pontes (Porto), Kirsten Linsmeier, Nicola Passow, Cornelia Rütz, Carmen Scherer (Heidelberg), Klaas Kooistra and Jelle Conradie (Groningen) for technical assistance. The BIOMED-2 Pathology Review Panel consisted of JHJM van Krieken (Nijmegen, coordinator), F Berger (Lyon), B Jasani (Cardiff), D Canioni (Paris), PhM Kluin (Groningen), JM Cabeçadas (Lisboa), TJ Molina (Paris), G Delsol (Toulouse), A Orfao (Salamanca), JF Garcia (Madrid), M Ott (Würzburg), P Gaulard (Creteil), ST Pals (Amsterdam) and ML Hannsmann (Frankfurt). This study was supported by the BIOMED-2 Concerted Action grant BMH4-CT98-3936 of the European Commission. TJM was supported by a ‘Ligue contre le cancer, Comité de Paris’ equipment grant. FLL was supported by a grant from the Leukaemia Research Fund, UK.
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Langerak, A., Molina, T., Lavender, F. 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, 222–229 (2007). https://doi.org/10.1038/sj.leu.2404482
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