Practical detection of t(14;18)(/IgH/BCL2)in follicular lymphoma.
The t(14;18)(q32;q21) translocation is the genetic hallmark of
follicular lymphoma. Detection of this translocation can facilitate the
diagnosis of follicular lymphoma and can be used to monitor response to
therapy and level of residual disease. We herein review and compare
practical techniques for detecting t(14 18)(q32 q21), including
conventional cytogenetics, fluorescence in situ hybridization, Southern
blot analysis, and polymerase chain reaction-based assay. Emphasis is
placed on fluorescence in situ hybridization and polymerase chain
reaction-based assay, given the applicability of these techniques to
fixed, paraffin-embedded tissue.
(Arch Pathol Lab Med. 2008;132:1355-1361)
Translocation (Genetics) (Identification and classification)
Polymerase chain reaction (Methods)
Chan, Wing C.
Hawley, Robert C.
|Publication:||Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165|
|Issue:||Date: August, 2008 Source Volume: 132 Source Issue: 8|
|Topic:||Event Code: 310 Science & research|
|Geographic:||Geographic Scope: United States Geographic Code: 1USA United States|
As summarized in the World Health Organization classification,
follicular lymphoma (FL) is a neoplasm of follicle center B cells that
makes up approximately 22% of non-Hodgkin lymphomas worldwide and about
35% of non-Hodgkin lymphomas in the United States. It primarily affects
adults (median age of onset, 59 years) and is rare in persons younger
than age 20. Patients often present with generalized disease involving
lymph nodes, spleen, bone marrow and, in some cases, peripheral blood.
Extranodal sites may be involved primarily or secondarily. Follicular
lymphoma is generally considered to be indolent but incurable, with a
median overall survival of 7 to 10 years. Transformation to aggressive
B-cell lymphoma occurs in about 30% of cases and is associated with poor
outcome. Follicular lymphoma has a predominantly follicular to focally
follicular pattern of infiltration, contains a variable admixture of
centrocytes and centroblasts, and can be histologically graded according
to the average number of centroblasts within follicles (0-5 centroblasts
per high-power field, grade 1; 6-15 per high-power field, grade 2; and
>15 per high-power field, grade 3). Follicular lymphoma cells
typically express B-cell markers (CD19, CD20, CD22, and CD79a), CD10,
BCL-6, BCL-2, and
monotypic surface immunoglobulin, and they have clonally rearranged, hypermutated immunoglobulin genes. (1)
The t(14;18)(q32;q21) translocation is the genetic hallmark of FL and is an early event in follicular lymphomagenesis. (2) This translocation can be identified in 85% to 90% of cases of nodal follicular lymphoma (3) andinupto 40% of cases of primary cutaneous follicle center cell lymphoma. (4) However, it is not entirely specific for FL, as it also can be detected in 20% to 30% of cases of de novo diffuse large B-cell lymphoma (5) and at a very low level in peripheral blood, bone marrow, and lymphoid tissues of a high proportion of people who have no evidence of lymphoma. The frequency of the translocation in healthy individuals increases with age and smoking. (6) Juxtaposition of the B-cell leukemia/lymphoma 2 gene (BCL2) at 18q21 and the immunoglobulin heavy chain gene (IgH) at 14q32 results in enhanced transcription of BCL2, overexpression of anti-apoptotic bcl-2 protein, a selective survival advantage, and, in conjunction with additional genetic alterations, progression to FL. (2) The breakpoints at 14q32 are in the joining region ([J.sub.H])of IgH in nearly all cases. Breakpoints at 18q21 occur in the major breakpoint region (MBR), a 150-bp segment within the 3' noncoding portion of BCL2 exon 3, in roughly 50% to 65% of cases; in the minor cluster region (mcr), a 500-bp segment 20 to 30 kb 3' to the MBR, in about 10% of cases; and in additional breakpoints/clusters between the MBR and the mcr (including the 3' BCL2, 3' MBR, intermediate cluster region, and 5' mcr) in most of the remaining cases (Figure 1, A through C). (7-11)
Detection of t(14;18) is not ordinarily required for diagnosis of FL but may be useful for confirmation of the diagnosis with small biopsies and atypical follicular lymphoid proliferations when routine histology, immunophenotyping, and/or immunoglobulin gene rearrangement analysis are inconclusive. (3) Testing for t(14;18) can also be used to monitor response to therapy, (12) detect recurrent disease,12 evaluate effectiveness of FL cell purging in autologous stem cell harvests, (12) and perhaps even define the biologic subtype of FL. (13) A subtype of FL lacking the t(14; 18)(q32;q21) translocation, characterized by low-intensity or absent bcl-2 expression and a tendency to occur in extranodal sites, particularly the skin, has a more favorable outcome than nodal t(14;18)(q32;q21)-positive FL. (13)
This article will review several well-established methods of detecting t(14;18)(q32;q21) including conventional cytogenetics, fluorescence in situ hybridization (FISH), Southern blot analysis, and polymerase chain reaction (PCR), with particular emphasis on interphase FISH and PCR, and will compare the relative strengths and limitations of each method.
[FIGURE 2 OMITTED]
METHODS OF DETECTING t(14;18)
Conventional Cytogenetic Analysis
Conventional cytogenetic analysis is rarely needed to establish a diagnosis of FL but, nonetheless, can detect t(14;18)(q32;q21) in diagnostic biopsies containing FL with a high degree of sensitivity. (14,15) When metaphases are obtained in sufficient number, t(14;18)(q32;q21) can be identified in approximately 85% to 90% of grade 1 and grade 2 FL and 75% of grade 3 FL. (2,15) Moreover, conventional cytogenetics can evaluate the entire karyotype and identify abnormalities such as +X, +7, +12, +12q, +18, 11p--, 16q--, 10q--, 13q--, and 17q--that may be present in addition to the t(14;18) and may be of prognostic significance. (2,15) Conventional cytogenetic study requires fresh material with viable dividing cells, and fresh lymph node biopsy material is not routinely submitted for cytogenetic study in many medical centers. Additional limitations are that conventional karyotyping cannot distinguish the t(14; 18)(q32;q21)-IgH/BCL2 found in FL from the t(14;18)(q32; q21)-IgH/MALT2 found in a subset of extranodal marginal zone lymphomas of mucosa-associated type, and it may not detect the t(14;18) when the cells that contain it have a low proliferative rate or are present in low numbers. Conventional karyotyping is also relatively laborious and expensive and typically requires several days to complete.
Fluorescence In Situ Hybridization
In contrast to conventional cytogenetic analysis, which can assess the whole karyotype, FISH, Southern blot analysis, and PCR analysis target specific genetic abnormalities. Like conventional cytogenetics, FISH can be performed on dividing cells (metaphase FISH). However, FISH also can be performed on nondividing cells (inter-phase FISH) using smears, cytospin preparations, touch imprints, frozen tissue, and fixed, paraffin-embedded material. Its application to fixed, paraffin-embedded tissue makes it particularly useful not only for diagnostic pathology but also for research using archival material. Formalin fixation may be superior to B5 fixation for FISH analysis. (16) Fluorescence in situ hybridization can be performed on either deparaffinized tissue sections or on whole nuclei extracted from paraffin-embedded tissue. (16,17) The former approach is technically simpler and preserves the histologic architecture, whereas the latter approach eliminates nuclear overlap and truncation and thereby facilitates interpretation. Multicolor FISH and spectral karyotyping can resolve complex karyotypic abnormalities and ambiguities in banding and components in marker chromosomes, but such high resolution is not ordinarily required for detection of t(14;18).
Fluorescence in situ hybridization detection of t(14; 18)(q32;q21) uses fluorescent DNA probes that bind specifically to select regions on one or both of the involved chromosomes. There are several different approaches to probe set design, including break-apart, single-fusion, and dual-fusion designs. The dual-color, dual-fusion technique is particularly well suited to the detection of t(14;18). (18,19) Dual-color, dual-fusion FISH uses 2 different colored DNA probes, 1 of which can essentially span the entire IgH gene and the other the entire BCL2 gene, and can detect translocations occurring at breakpoints spanned by the probes (Figure 1, A and B). The dual-color, dual-fusion technique yields 1 normal IgH signal, 1 normal BCL2 signal, and 2 fusion signals (1 from each of the derivative chromosomes) when t(14;18) is present (Figure 2, A and B). Dual-color, dual-fusion FISH is less prone to spurious signal patterns arising from DNA degradation than are break-apart techniques. It is also less subject to false-positive patterns stemming from coincidental colocalization of signals in normal background cells than are single fusion techniques. (18,19)
Dual-color, dual-fusion FISH can detect t(14;18) in 64% to 100% of follicular lymphomas. (16-22) False-positive results can be minimized by setting the positive/negative threshold at a high enough level (typically at the mean for normal cells + 3 SD). With a fairly conservative threshold (eg, about 5%), dual-color, dual-fusion interphase FISH can detect as few as 5 cells with t(14;18) in a background of 100 normal cells (5% analytic sensitivity). (16-22) This level of analytic sensitivity is generally adequate for diagnosis of FL at time of initial diagnosis or relapse when the number of lymphoma cells is relatively large, but it is not sufficient for detection of minimal residual disease. Polymerase chain reaction--based assays are required for reliable detection of minimal residual disease. However, dual colordual fusion FISH has a higher overall diagnostic sensitivity than standard PCR because the FISH probes span almost all breakpoints, whereas standard PCR primer sets bracket only a limited number of breakpoint regions. (20-22) In addition, FISH probes do not require absolute sequence complementarity and are not as adversely impacted by poor quality of specimen DNA. Fluorescence in situ hybridization also can detect, but not definitively characterize, certain complex cytogenetic abnormalities and translocations other than IgH/BCL2 involving the IgH or BCL2 genes, such as +der(18)t(14;18)(q32;q21), t(2;18)(p11;q21), and t(18;22)(q21;q11). (22) Fluorescence in situ hybridization is more expensive to perform than PCR analysis but is no more costly than conventional cytogenetic analysis. The analytic and diagnostic sensitivity of FISH is slightly superior to that of conventional cytogenetics because more cells are evaluated in FISH analysis. In addition, FISH does not require dividing cells. Fluorescence in situ hybridization scoring is time consuming but has the potential to be automated. (16) Average turnaround time for FISH analysis is 2 to 3 days.
Southern Blot Analysis
Southern blot analysis using probes for MBR and mcr can detect translocations involving the BCL2 gene in approximately 75% of FL and has an analytic sensitivity of 1% to 5%. (14) However, traditional Southern blot analysis requires fresh or frozen material and a relatively large amount of high molecular weight DNA (5-10 [micro]g per digest) and, with restriction endonuclease digestion, electrophoresis, transfer, and probing, is relatively laborious and slow (1-2-week turnaround time). Moreover, traditional Southern blot analysis requires radiolabeled probes for highest sensitivity. Given these limitations, FISH and PCR-based techniques have essentially supplanted Southern blot analysis for detection of t(14;18) and other lymphoma-related translocations in the diagnostic clinical laboratory.
[FIGURE 3 OMITTED]
Standard PCR.--Given its relative simplicity, PCR analysis is well-suited for use in the diagnostic molecular laboratory. Polymerase chain reaction can be effectively employed to detect t(14;18)(IgH/BCL2) because the chromosomal breakpoints at 14q32 are tightly clustered in the [J.sub.H] region of the IgH gene, and the majority of those at 18q21 are clustered in either the MBR or the mcr of the BCL2 gene. (7-10) However, standard PCR is much less effective than FISH for detection of translocations such as the t(11; 14)(BCL1 /IgH) in mantle cell lymphoma and the t(8; 14)(MYC/IgH) in Burkitt lymphoma because the breakpoints in BCL1 in mantle cell lymphoma and MYC and IgH in Burkitt lymphoma are heterogeneous.
Polymerase chain reaction--based analysis can be performed on fresh, frozen, or fixed paraffin-embedded material and can be completed in several hours. Fresh and frozen material are more suitable than fixed material, and formalin fixation is superior to B5 fixation for PCR analysis. (20-22) Because PCR exponentially amplifies DNA target sequences, only a small amount of DNA (about 100 ng) is required initially. The amplification process can include multiple primer sets (multiplex PCR), each designed to amplify a different target, and thereby screen for different BCL2 breakpoints in a single reaction. Gel electrophoresis has been commonly employed to detect the amplification product (Figure 3, A and B). However, use of fluorescent primers with detection by capillary electrophoresis is becoming increasingly popular in the diagnostic clinical laboratory arena (Figure 4, A and B). Polymerase chain reaction testing is not well standardized. (23,24) In one effort to address this problem, a European collaborative group (BIOMED group) (25) has developed a number of standard ized PCR protocols, including a protocol for detection of t(14;18)(IgH/BCL2). Polymerase chain reaction assay using MBR/[J.sub.H] and mcr/[J.sub.H] primer sets can detect the translocation in up to 64% of FL in fixed tissue and up to 70% in frozen tissue. (14,22,26,27) However, PCR will not detect translocations with breakpoints outside the regions bracketed by the primer sets. The multiplex PCR strategy described in the BIOMED-2 report using primer sets capable of detecting breakpoints in the MBR, mcr, and 2 specific regions between the MBR and mcr (3' MBR and 5' mcr) can detect the translocation in up to 88% of FL when frozen tissue is used for analysis. (20,21,25) Long-range PCR techniques can amplify long segments of DNA (2 kb to up to 16-23 kb, as opposed to 100-1000 bp in standard PCR) and detect most breakpoints (7,8) but require high-quality DNA from fresh or frozen tissue and are technically more demanding. Standard, single-round PCR is sensitive enough to detect roughly 1 FL cell in a background of 1000 to 10 000 nonlymphomatous cells. (23,25) Nested PCR in which an initial round of conventional PCR is followed by a second round of amplification with primers that anneal at sites internal to the initial set can be 100-fold more sensitive, but it is prone to false-positive results stemming from contamination. (23)
Quantitative PCR.--Standard qualitative PCR is sufficient for initial diagnosis of FL; however, more precise quantitation is needed for evaluation of minimal residual disease. Real-time PCR is an excellent means to quantitate the level of residual disease, provided the BCL2 breakpoint is one that can be amplified by PCR. Real-time PCR also can be used to detect t(14;18) at initial diagnosis and has a relatively rapid turnaround time because amplification and detection are accomplished in the same phase. Real-time PCR uses a fluorogenic probe or dye to measure the accumulation of PCR product in real time. The product accumulation rate and corresponding change in fluorescence intensity are dependent upon the number of IgH/ BCL2 fusion sequences initially present (Figure 5, A). If SYBR green intercalating dye is used, dissociation (melting) curve analysis can be performed on the PCR product to verify that it is the intended product (Figure 5, B). Real-time PCR requires only a minimal amount of DNA (0.1-20 ng), no postamplification detection steps, and no more than several hours to complete. In addition, the analysis is done in a closed system, which, together with elimination of post-PCR manipulation, minimizes the risk of contamination. The role of real-time PCR in the management of patients with follicular lymphoma is not clearly defined. Nevertheless, real-time PCR can be performed on bone marrow or peripheral blood at the time of diagnosis to assess the level of IgH/BCL2 and during and after treatment to monitor response to therapy and to detect recurrence. (12) Real-time PCR also can be performed on harvested autologous stem cells to assess the efficacy of FL cell purging. (12) Analytic sensitivity is on the order of [10.sup.-4] to [10.sup.-5] (12,28,29) Quantitative PCR is not well standardized between laboratories, and comparison of results obtained in different laboratories may be difficult. (24)
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
Given that t(14;18) can be identified at a low level in many people without FL, a low level of t(14;18) in a patient on therapy for FL may not always reflect the presence of malignant cells. (29) However, interval quantitation of IgH/ BCL2 fusion sequences should allow distinction between relapsing FL and a benign clone; an increasing level of IgH/BCL2 would be in keeping with relapse. Alternatively, size or sequence analysis of quantitative PCR product could be done to confirm identity with the neoplastic clone. (12) Quantitative PCR can be coupled secondarily with a technique such as high-resolution capillary electrophoresis to assess amplicon size. (30)
Detection of t(14;18) in the Initial Diagnosis of FL
Recent clinical practice guidelines of the National Comprehensive Cancer Network indicate that cytogenetic study and molecular analysis are not essential for the initial diagnosis and workup of FL but can be useful in selected circumstances. (31) Detection of t(14;18) may be particularly useful in the context of equivocal morphologic and immunophenotypic findings. Fluorescence in situ hybridization and standard PCR currently have the broadest application in this context. Interphase FISH and PCR are complementary in many respects, and either technique can be effectively used in the initial diagnosis of FL. Fluorescence in situ hybridization has greater diagnostic sensitivity than PCR, particularly with fixed, paraffin-embedded tissue (Table 1). (19-22,32) Fluorescence in situ hybridization probes that span the entire IgH and BCL2 genes can detect translocations arising at nearly all breakpoint sites and do not require absolute sequence complementarity, whereas PCR primers can detect breakpoints only within targeted regions and are more adversely affected by degradation of specimen DNA. On the other hand, FISH may miss focal involvement by lymphoma. Polymerase chain reaction analysis is faster and less expensive to perform than FISH and has greater analytic sensitivity than FISH; however, high analytic sensitivity is not ordinarily required at the time of initial diagnosis, when lymphoma cells tend to be abundant. Somatic hypermutation of IgH may reduce the detection rate of t(14;18) by PCR. However, somatic hypermutation also reduces the PCR detection rate of clonal IgH rearrangements in FL, and t(14;18) testing may be more sensitive than immunoglobulin gene rearrangement studies for detecting FL in some cases. Features of FISH and standard PCR are compared in Table 2. The presence of a benign t(14;18) in reactive lymphoid tissues generally does not present a problem at the time of initial diagnosis with either FISH or standard PCR, because the benign translocation usually is present at a very low level, below that of detection by FISH or even standard PCR. Given its superior diagnostic sensitivity, FISH could readily be employed as the front-line molecular test for t(14;18) when such testing is indicated. Alternatively, it has been suggested that PCR, the less expensive and faster technique, could be used initially and that FISH could be used secondarily for PCR-negative cases. (20)
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(31.) NCCN clinical practice guidelines in oncology, non-Hodgkin's lymphomas v.3.2007. Available at: http://www.nccn.org. Accessed November 2007.
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Keni Gu, MD, PhD; Wing C. Chan, MD; Robert C. Hawley, MD
Accepted for publication January 30, 2008.
From the Department of Pathology, Henry Ford Hospital, Detroit, Mich (Drs Gu and Hawley); and the Department of Pathology and Microbiology (Drs Gu and Chan) and Center for Lymphoma and Leukemia Research (Dr Chan), University of Nebraska Medical Center, Omaha.
The authors have no relevant financial interest in the products or companies described in this article.
Research was done by Dr Gu at Henry Ford Hospital, Detroit, Mich, and continued at his new and current affiliation, University of Nebraska Medical Center, Omaha.
Reprints: Robert C. Hawley, MD, Department of Pathology, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI 48202 (e-mail: firstname.lastname@example.org).
Table 1. Results of Studies Directly Comparing Dual Color-Dual Fusion Interphase Fluorescence In Situ Hybridizatio (FISH) and Standard Polymerase Chain Reaction (PCR) for Detection of t(14;18) in Follicular Lymphoma * FISH Material Positive Cases, Study Group Preparation No./Total (%) Godon (19) Frozen 39/40 (98) Barrans (20) Frozen 24/26 (92) Paraffin 26/28 (93) Einerson (22) Paraffin 24/28 (86) Gu (32) Paraffin 38/50 (76) Belaud-Rotureau (21) Frozen Paraffin 25/28 (89) 37/41 (90) PCR Study Group BCL2 Target Godon (19) MBR Barrans (20) MBR, mcr, 3' MBR, 5' mcr Einerson (22) MBR, mcr Gu (32) MBR, mcr Belaud-Rotureau (21) MBR, mcr, 3' MBR, 5' mcr PCR Material Positive Cases, Study Group Preparation No./Total (%) Godon (19) Frozen 19/33 (58) Barrans (20) Frozen 23/28 (82) Paraffin 8/20 (40) Einerson (22) Paraffin 5/28 (18) Gu (32) Paraffin 32/50 (64) Belaud-Rotureau (21) Frozen Paraffin 20/28 (71) 25/51 (49) * BCL2 indicates B/cell leukemia/lymphoma 2 gene; MBR, major breakpoint region; mcr, minor cluster region. Table 2. Comparison of Several Key Features of Conventional Cytogenetics, Interphase Fluorescence In Situ Hybridization (FISH), and Standard Polymerase Chain Reaction (PCR) (Approximate Values) Analytic Method Specimen Sensitivity Cytogenetics Fresh [10.sup.-1] FISH Fresh, frozen, fixed [10.sup.-1]-[10.sup.-2] PCR Fresh, frozen, fixed [10.sup.-3]-[10.sup.-4] Diagnostic Turnaround Method Sensitivity, % Time Cost * Cytogenetics 85 1 wk $1500 FISH 65-100 2 d $500-1000 PCR 65-70 4 h $250-500 * These costs are estimates; actual charges for laboratory testing vary based on facility and client, and possible annual cost increases.
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