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Received: October 30^th 2013
Accepted: May 29^th 2014

Clonality lymphoid study through rearrangement analysis of antigen receptor

Nicolás Villamizar-Rivera,^a Natalia Olaya^a

^aGrupo de Patología Oncológica, Instituto Nacional de Cancerología-ESE, Bogotá, Colombia

Communication with: Natalia Olaya
Telephone: (1) 334 11 11, extension 4222
Email: nolaya@cancer.gov.co

As a rule, malignant lymphoid proliferations are clonal. While most of the time the biological potential can be established through routine pathologic examination and auxiliary techniques, some cases are difficult to classify. Moreover, there are situations in which there are dominant clones whose analysis are important, such as occur in autoimmune diseases and immunodeficiency. This paper presents in an understandable way the main techniques for the study of clonality in lymphoid lesions, i.e. the analysis of rearrangements of antigen receptor genes by multiplex polymerase chain reaction (PCR) based tests.

Keywords: DNA rearrangements; Lymphoma; Leukemia; Neoplasm; Clonal evolution

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The current theory to explain the development of cancer is evolution. Some somatic cells are selected and prevail despite the struggle for space and nutrients and the control mechanisms of cell division and aging.¹ The existence of dominant clones usually defines the disease.

Leukemias and lymphomas are cancers arising from lymphocytes or their parents; they affect any bodily topography and people of all ages. To diagnose and classify them, morphology, immunophenotype, and certain genetic alterations are evaluated.² In mature lymphoid proliferations, the ratio of expression of immunoglobulin λ and κ chains is determined.³

These methods can classify the most lesions, but in difficult cases patients experience delayed treatment or undergo inappropriate procedures. The number of lesions of uncertain classification varies according to the experience of the diagnosis center. In addition, it is sometimes necessary to establish the clonal relationship between two lesions or follow up after treatment.⁴

The rearrangement of antigen receptor genes is a physiological process whose study can help determine lymphoid clonality. Candidates for this analysis include proliferations of B cells that are difficult to classify, all T cell lesions, and those occurring in transplant patients, immunocompromised patients, those with autoimmune diseases or diseases associated with lymphocyte proliferation, such as inflammatory bowel disease and celiac disease.^5,6

We intend to introduce readers to the scientific principles and application of the technique to study gene rearrangements of antigen by polymerase chain reaction (PCR).

Rearrangement of antigen receptor gene

The genes of immunoglobulins (Ig) and the T cell receptor (TCR) are present in all cells of the body, but they are like an unassembled puzzle. The loci encoding the heavy chains of Ig, the light chains κ and λ of Ig (Figure 1A), and the α, β, γ, and δ chains of the T cell receptor (TCR) are on different chromosomes (Figure 1B). In addition, there are several possible sequences for each of the segments constituting the variable region of the gene. For these reasons, the organization of each germline gene cannot be transcribed in ARNm.⁷

Only lymphocytes require converting gene fragment sections into functional genes for antigen receptors, thereby generating antigenic diversity. In each lymphocyte, somatic recombination produces an exon that encodes the variable region based on the selection of a variable segment V, a linking segment L, and a diversity segment D, and the addition of nucleotides N and P. Segment D is only present in the loci of the Ig heavy chains, and β and δ loci are unique to TCR (Figure 2). VDJ allelic recombination is controlled by allelic exclusion; once an allele has been rearranged, a signal is sent to the other to stop the process.^7,8

Rearrangements are performed through sequential and unchanging steps:

1. Synapse: the enzymatic mechanism detects recombination signal sequences or RSS. These consist of three parts: a heptamer of conserved nucleotides, a spacer of 12 or 23 variable nucleotides, and a nonamer of nine AT-rich nucleotides. The heptamers are vulnerable to VDJ recombinase, a tetramer consisting of RAG 1 and 2 proteins. These enzymes are specific to developing lymphocytes and are inactive during cell proliferation. 
2. Excision: these are double-strand breaks in DNA at the joints between the RSS and the coding sequence. The connection between the heptamers is accomplished by removing the DNA between them, or forming a loop by reversing the chains. The broken encoding ends end in a closed fork.⁹
3. Opening of the forks through Artemis enzyme, which is an endonuclease, so that the TdT enzyme adds new bases in the exposed ends, increasing the diversity of sequences. 
4. Union and completion through the system of non-homologous end joining, a DNA repair system present in every cell.

Later, mature B lymphocytes increase their combinatorial repertoire through somatic hypermutation, which occurs in the germinal center. This is characterized by the addition or subtraction of nucleotides.⁸

It is still unknown how a particular locus is selected in a specific case or why some fragments are chosen more often. In lymphoid neoplasms, rearrangements of one receiver or another happen regardless of lesion lineage, but during the physiological process variable regions of Ig are assembled in B lymphocytes, whereas the variable regions of TCR are coupled only in T lymphocytes.

Technical principles

Southern blotting was used for a long time. The technique is very specific but laborious, and the demands limit the application, as it requires about 20 ug high-quality DNA.¹⁰ For ten years there has been group of protocols based on PCR, called BIOMED-2.⁴ These were developed by a group of European laboratories, which later formed the consortium EuroClonality;¹¹ some of the necessary materials are available from commercial companies.¹² Numerous publications have appeared about their performance and others that provide improvements on the original technique.^13,14

TCR rearrangements, particularly the β TCR repertoire, can be assessed by flow cytometry (FC).¹⁵ The Euroflow consortium proposes a panel of 24 antibodies, which determine 70% of domains.¹⁶ FC also allows quick quantitative assessment and follow-up.³

The study of gene rearrangements of the antigen receptor requires a multidisciplinary exercise between pathology, FC, genetics, and molecular biology.¹⁷

Technical procedure

Samples

Technical protocols are designed for use with fresh or frozen tissue. However, they have been successful in paraffin-treated tissues.^18,19 Moreover, FC enables the use of blood, bone marrow, and other fluids. For any tissue, the condition is that it contain a representative number of problem cells.^4,16

The pathologist will choose the area of ​​tissue to be examined. If this is the case, they will perform macro or microdissection of the sample in order to increase problem cells, but it is important that polyclonal populations persist. Naturally oligoclonal regions should be avoided, such as the germinal centers of lymphoid follicles.

Negative controls are polyclonal populations of lymphocytes obtained from normal peripheral blood or lymphoid tissues. Commercial cell lines with known rearrangements or characterized clonal lesions are used as positive controls.^4,20

To facilitate interpretation, it is necessary to have information on the case study: demographic and clinical data, pathology report, immunophenotype, presence and amount of reactive lymphocytes, et cetera. Treatments received affect the results as well; for example, administration of anti-CD20 makes it difficult to determine clonality of B cells, and some translocations such as t (11; 14) and t (14; 18) may give false negative results because of involvement of aberrant IgH rearrangements.²¹

Nucleic acid isolation and quality assessment

Ischemia time, procedures for freezing and storage, and inclusion of paraffin affect the quality of the DNA extracted from tissues. The type and the time of fixation, tissue thickness, DNA extraction procedures, and the presence of PCR inhibitors are all important.¹⁹

DNA tests require high purity and low fragmentation. If fresh or frozen samples are used, the traditional and commercial extraction methods work well, including using automatic extractors. In the case of tissue in paraffin, it is recommended to use 10% buffered formalin as fixative and careful control of setting times. Although the BIOMED-2 protocols recommend the QIAmp DNA Mini Kit (QIAGEN®), other publications suggest organic extraction and purification.^18,22 In any case, if tissue in paraffin is used, it is recommended to check that it is amplifiable to at least 300 nucleotides. BIOMED-2 includes an amplification protocol that allows evaluating products of various sizes, but there are other options.⁴

Target selection

The selection of the fragments to be amplified depends on the clinical pathological question and the amount and quality of extracted DNA. In the case of paraffin-treated tissue, the smallest amplicons must almost always be evaluated. In addition, the clinical question helps, since the frequency and distribution of rearrangements varies with tumor taxonomy (Table I).

If the amount of lymphoid cells in the sample is low, as in skin or intestine infiltrations, it is necessary to adjust the amounts of DNA and duplicate testing. An algorithm for the selection of molecular targets is proposed (Figure 3).

Amplification and analysis of amplicons

It is not possible to detect all possible gene segments by PCR, as that requires too many primers and tubes. For this reason, groups of multiplex PCR were designed using primers that recognize most of them and consensus primers that recognize conserved sequences among the fragments. No special thermocycler is needed.^4,16

Analysis of the products of the PCR multiplex is done by means of acrylamide or capillary gel electrophoresis. High-resolution microcapillary electrophoresis has also been done using Agilent 2100 bioanalyzer equipment.²³ Double-stranded fragment analysis can be done (heteroduplex formation) as well as analysis of products in a single chain. In order to induce heteroduplex formation, multiplex PCR products are subjected to rapid heating and cooling.²⁴ The presence of a dominant peak or band indicates positivity for the rearrangement studied, and it indirectly suggests the presence of a clone (Figure 4).⁴

Interpretation and difficulties

It is necessary to know the structure of the genes to be evaluated, their reordering patterns, and variations of these; one must also verify the expression patterns of the controls (Table II). The technique should not be used to determine the lineage of the lesion. Furthermore, experimental results should be reproducible.²⁵

False positives or negatives happen in the implementation of these tests. Many of them can be avoided through careful analysis. For example, the evaluator may face PCR products that do not comply with the expected size, or multiple products. For samples containing few lymphocytes, unique nonspecific peaks may be obtained, which is called pseudoclonality. In addition, tissues, including tumor tissues, usually have a normal polyclonal basis, which, if they are very prominent, it may be difficult to interpret.

False negatives occur for several reasons:

1. Consensus primers or initiators were used; in this case, about 80% of possible segments are amplified, but not all.
2. In mature B-cell malignancies of the germinal center, whose cells may have undergone somatic hypermutation, there will be no amplification.
3. The lesion is underrepresented in the tissue examined.⁴

The detection of one or more clones does not imply the presence of a malignancy, which is why we insist that the results should be interpreted in conjunction with all disciplines related to the patient.17 EuroClonality services may be used for interpreting confusing results.¹¹
While in Mexico and Latin America some groups are experienced with the use of these techniques, this is still relatively marginal.^26-29

Minimal residual disease and rearrangements

Current treatments do not eradicate malignancies, but reduce the number of tumor cells.³⁰ In order to predict and prevent clinical relapse, it is necessary to detect blasts in subclinical stages, which is known as minimal residual disease (MRD). This is an important prognostic factor in leukemias and lymphomas. It serves to monitor and to determine the need for trasplant.^31-33

The purpose of the tests for determining MRD is to detect even the last blast (Table II). When the disease is associated with a specific genetic damage, this can be used as a marker of MRD. However, in some cases, such as TEL-AML1 transcript, most patients develop molecular remission after induction; on the contrary, in the case of BCR-ABL, most patients remain positive after induction.³⁴

For leukemias, immunophenotyping using FC and molecular biology techniques for determining genes rearrangements of antigen receptors provide high sensitivity. To determine MRD through tests for Ig and TCR rearrangements, protocols are applied to a diagnostic sample rich in tumor cells. The most representative cells are sequenced. Based on the results, specific primers are designed and quantitative trials for PCR are put in place.³⁵ 30% of cases have clonal evolution and require monitoring for several rearrangements.³¹ However, it can be said that tracking through this technique is not only very precise but customized.

Conclusions

Although procedures to confirm lymphoid clonality through the search for rearrangements of antigen receptor genes are known and have been used for decades, their use must be extended and exchanges must be established between disciplines in order to improve patient care.

References

1. Greaves M, Maley CC. Clonal evolution in cancer. Nature 2012;481(10762):306-13.
2. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Fourth Edition, Lyon, France: WHO; 2008.
3. Jaffe E, Lee-Harris N, Vardiman J, Campo E, Arber DA.
4. Van Dongen JJM, Langerak AW, Brüggemann M, Evans PAS, Hummel M, Lavender Fl, et al. Design and standarization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia. 2003;17:2257-317.
5. Perfetti V, Brunetti L, Biagi F, Ciccocioppo R, Bianchi PI, Corazza GR. TCRβ clonality improves diagnostic yield of TCRγ clonality in refractory celiac disease. J Clin Gastroenterol. 2012;46(8):675-9.
6. Sirsath NT, Channaviriappa L, Nagendrappa LK, Dasappa L, Sathyanarayanan V, Setty GB. Human immunodeficiency virus – associated lymphomas: a neglected domain. N Am J Med Sci. 2013;5(7):432-7.
7. Abbas AK. Inmunología celular y molecular. Ninth edition: San Francisco, USA: El Sevier; 2009.
8. Giallourakis CC, Franklin A, Guo C, Cheng HL, Yoon HS, Gallagher M, et al. Elements between the IgH variable (V) and diversity (D) clusters influence antisense transcription and lineage-specificV(D)J recombination. Proc Natl Acad Sci USA. 2010;107(51):22207-12.
9. Deriano L, Chaumeil J, Coussen M, Multani A, Chou YF, Alekseyenko AV, et al. The RAG2 C-terminus suppresses genomic instability and lymphomagenesis. Nature. 2011;471(7336):119-23.
10. Knowles II DM, Neri A, Pelicci PG, Burke JS, Wu A, Winberg CD, et al. Immunoglobulin and T-cell receptor B-chain gene rearrangement analysys of Hodgkin’s Disease: Implications for lone age determination and differential diagnosis. Proc Natl Acad Sci USA. 1986;83(1):7942-6.
11. EuroClonality. EuroClonality 2013 [Cited 2013, Oct 10]. Availabre from http://www.euroclonality.org/
12. Invivoscribe. Invivoscribe | Personalized Molecular Medicine 20. [Cited 2013, Oct 10). Availabre from http://www.invivoscribe.com/
13. Melotti CZ, Carriel MF, Sotto MN, Diss T, Sanches JA. Polymerase Chain Reaction-Based Clonality Analysis of Cutaneous B-Cell Lymphoproliferative Processes. Clinics. 2010;65(1):53-60.
14. Harris S, Bruggemann M, Groenen PJTA, Shuuring E, Langerak AW, Hodges E. Clonality analysis in lymphoproliferative disease using the BIOMED-2 multiplex PCR protocols: experience from the EuroClonality group EQA scheme. Journal of Hematopathology. 2012;5(1-2):91-8.
15. Van den Beemd R, Boor PP, Van Lochem EG, Hop WC, Langerak AW, Wolvers-Tettero IL, et al. Flow cytometric analysis of the Vbeta repertoire in healthy controls. Cytometry. 2000;40(4):336-45.
16. Kalina T, Flores-Montero J, Van der Velden VH, Martin-Ayuso M, Böttcher S, Ritgen M, et al; EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708). EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols. Leukemia. 2012;26(9):1986-2010.
17. Van Krieken JH, Cabecadas J, Groenen PJ. Clonality testing: teamwork by pathologist and molecular biologist. J Hematopathol 2012;5(1-2):3-5.
18. Bonzheim I, Frölich F, Adam P, Colak S, Metzler G, Fend F, et al. A comparative analysis of protocols for detection of T cell clonality in formalin-fixed, paraffin-embeddestissue- implications for practical use. Journal of Hematopathology. 2012;5(1-2):7-16.
19. Paireder S, Werner B, Bailer J, Werther W, Schmid E, Patzak B, et al. Comparison of protocols for DNA extraction from long-term preserved formal infixed tissues. Anal Biochem. 2013;2(439):452-60.
20. Van Krieken JH, Langerak AW, Macintyre EA, Kneba M, Hodges E, Sanz RG, et al. Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia. 2007;21(2):201-6.
21. Medeiros LJ, Carr J. Overview of the role of molecular methods in the diagnosis of malignant lymphomas. Arch Pathol Lab Med. 1999;123(12):1189-207.
22. Hebeda KM, Van Altena MC, Rombout P, Van Krieken JHJM, Groenen PJTA. PCR clonality detection in Hodgkin lymphoma. J Hematopathol 2013:34-41.
23. Beaufils N, Ben Lassoued A, Essaydi A, Dales JP, Formisano-Tréziny C, Bonnet N, et al. Analysis of T-cell receptor-γ gene rearrangements using heteroduplex analysis by high-resolution microcapillary electrophoresis. Leuk Res. 2012;36(9):1119-23.
24. Evans PA, Pott Ch, Groenen PJ, Salles G, Davi F, Berger F, 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. 2007;21(2):207-14.
25. Langerak AW, Groenen PJ, Brüggemann M, Beldjord K, Bellan C, Bonello L, et al. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lyphoproliferations. Leukemia. 2012;26(10):2159-71.
26. Ortiz YM, Arias L, Alvarez CM, García LF. Memory phenotype and polyfunctional T cells in kidney transplant patients. Transpl Immunol 2013;28(3):127-37.
27. Piña-Oviedo S, Fend F, Kramer M, Fournier F, Farca A, Ortiz-Hidalgo C. Diagnosis of early gastric marginal zone lymphoma (MALT lymphoma) in endoscopic biopsies. Report of a case that demonstrates the utility of immunohistochemistry and the molecular analysis. Rev Gastroenterol Mex. 2008;73(3):172-6.
28. Stefanoff CG, Hassan R, Gonzalez AC, Andrade LA, Tabak DG, Romano S, et al. Diagn Mol Pathol. 2003;12(2):79-87.
29. Bosaleh A, Denninghoff V, García A, Rescia C, Avagnina A, Elsner B. Rearreglos de genes de cadenas pesadas de las inmunoglobulinas en las gammapatias monoclonales.
30. Quesnel B, Preudhomme C. Residual disease: the hematologist’s point of view. Bull Cancer. 2001;88(6):571-5.
31. Garand R, Beldjord K, Cavé H, Fossat C, Arnoux I, Asnafi V, et al. Flowcytometry and IG/TCR quantitative PCR for minimal residual disease quantification in acute lymphoblastic leukemia: a French multicenter prospective study on behalf of the FRALLE, EORTC and GRAALL. Leukemia 2013;27(2):370-6.
32. Van Dongen JJ, Seriu T, Panzer-Grümayer ER, Biondi A, Pongers-Willemse MJ, Corral L, et al. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet. 1998;352(9142):1731-8.
33. Cavé H, Van der Werff ten Bosch J, Suciu S, Guidal C, Waterkeyn C, Otten J, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer—Childhood Leukemia Cooperative Group. N Engl J Med. 1998;339(9):591-8.
34. Weng XQ, Shen Y, Sheng Y, Chen B, Wang JH, Li JM. Prognostic significance of monitoring leukemia-associated immuniphenotypes by eight-color flowcytometry in adult B-acute lymphoblastic leukemia. Blood Cancer J. 2013;16(3):133-43.
35. Brüggemann M, Schrauder A, Raff T, Pfeifer H, Dworzak M, Ottmann OG, et al. Estandardized MRD quantification in European ALL trials: proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. Leukemia 2010;24(3):521-535.

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