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JMD 2004, Vol. 6, No. 4
Copyright © 2004 American Society for Investigative Pathology & Association for Molecular Pathology

Quantitative PCR Detection of t(14;18) bcl-2/JH Fusion Sequences in Follicular Lymphoma Patients

Comparison of Peripheral Blood and Bone Marrow Aspirate Samples

Alex Bowman, Dan Jones, L. Jeffrey Medeiros and Rajyalakshmi Luthra

From the Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In patients with follicular lymphoma (FL), it is unresolved whether peripheral blood (PB) can replace bone marrow (BM) aspirate samples for detection of bcl-2/JH fusion sequences that result from the t(14;18)(q32;q21). We compare here the results of quantitative polymerase chain reaction (q-PCR) analysis for bcl-2/JH involving the major breakpoint cluster region (mbr) on paired PB and BM aspirate samples from 60 consecutive FL patients. There was a significant correlation between the level of bcl-2/JH fusion sequence obtained from PB and BM aspirate samples (r = 0.886), with 82% of samples showing less than one log of difference. Patients who had histological evidence of FL involving concurrent BM biopsy specimens had moderate to high levels of bcl-2/JH in both PB and BM aspirate samples, allowing unequivocal determination of translocation status (median bcl-2/JH to cyclophilin level was 8.014%). In contrast, patients with no detectable FL in their BM biopsy specimens often showed low levels of bcl-2/JH in both PB and BM aspirate samples (bcl-2/JH to cyclophilin median level = 0.006%), in a range similar to background levels that could be detected in patients without FL (n = 15, median bcl-2 mbr/JH to cyclophilin level = 0.002%). We conclude that PB can be used in place of BM aspirate samples to test for the presence of bcl-2 mbr/JH fusion sequence in FL patients and that either PB or BM aspirate testing yields a rough approximation of the degree of BM involvement by FL. However, in patients with minimal levels of bcl-2/JH in PB or BM aspirate samples, confirmation of this result by testing the primary tumor is recommended to confirm the presence of an identical bcl-2/JH fusion sequence and exclude false-positive results.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Most cases of follicular lymphoma (FL) carry the t(14;18)(q32;q21), which places the bcl-2 gene at 18q21 adjacent to enhancer sequences of the immunoglobulin heavy chain (IgH) locus at 14q32, resulting in overexpression of bcl-2.1, 2, 3 Using conventional cytogenetics, the t(14;18) can be detected in approximately 85% of FL cases,4 with the majority of translocations occurring at either the major breakpoint cluster region (mbr) or the minor breakpoint cluster region (mcr). Using polymerase chain reaction (PCR) analysis, bcl-2/JH involving the mbr and mcr regions can be detected in 60 to 70% and 8 to 15% of FL cases, respectively.5, 6

The failure to detect the t(14;18) in 10 to 25% of FL cases can be due to use of other oncogenic pathways (ie, bcl-2-negative FL), methodological issues, or the presence of the t(14;18) involving breakpoints outside the mbr and mcr.7 The sensitivity of detection of the t(14;18) by any methodology, including PCR, also varies according to the extent of involvement by FL in the tested sample. While most FL patients have tumor cells present in bone marrow (BM) and peripheral blood (PB), the degree of involvement by lymphoma at these sites can often be low. Furthermore, BM aspirate samples frequently under-represent the extent of involvement because paratrabecular FL cells have a tendency to not be aspirated. Furthermore, the t(14;18) can be detected by PCR with primers specific for the mbr or mcr in patients without FL. These false-positive cases are attributed to bcl-2 mbr/JH fusion sequences that arise from benign lymphocytes, or preneoplastic lymphocytes that carry occult translocations.8, 9, 10, 11, 12

When PCR detection of the t(14;18) is used to monitor tumor burden, relapse, or minimal residual disease in FL patients,12, 13, 14, 15 the choice of samples to best avoid false-positive and false-negative results becomes critical. One study has shown that analysis of PB specimens is less predictive of relapse than analysis of BM aspirate specimens.13 By contrast, other studies have demonstrated that PB and BM aspirate samples usually yield similar results.14 The recent development of t(14;18) assays using real-time quantitative polymerase chain reaction (q-PCR) methods,15, 16, 17, 18, 19, 20, 21, 22 followed by fluorescent-based high-resolution capillary electrophoresis,23 has allowed for more accurate quantitation and size determination of bcl-2/JH fusion sequences. However, the detection sensitivity for bcl-2/JH in BM aspirate and PB samples using q-PCR methods has not been systematically addressed.

Here, we compare the levels of bcl-2 mbr/JH fusion sequences detected by q-PCR in concurrent PB and BM aspirate samples from 60 patients with FL known to have bcl-2/JH. For patients with moderate to high levels of bcl-2/JH, the results of q-PCR analysis of PB and BM aspirate samples correlates well, and thus PB can be used instead of BM aspirate material for this purpose. Most of the patients with high levels of bcl-2/JH also had histological involvement of the BM biopsy specimen by FL, and thus PB levels are an indication of systemic tumor dissemination. However, for patients with low levels of bcl-2/JH, most of whom do not have histological evidence of BM involvement by FL, the t(14;18) may or may not be derived from neoplastic cells, and thus confirmation by analysis of the primary tumor is suggested.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Group
Quantitative PCR analysis for the t(14;18) involving the mbr and mcr is routinely performed on PB and/or BM aspirate samples of all FL patients at time of presentation to our institution. In this study, we focused on 60 FL consecutive patients accessioned between 1999 to 2003 who had bcl-2 mbr/JH fusion sequences and who had PCR data from paired PB and BM aspirate samples. To establish the range of detection of bcl-2/JH levels that occur among patients without FL, we also included PB and BM aspirate samples from 15 patients diagnosed with other B-cell lymphoproliferative disorders who had detectable bcl-2/JH levels.

The primary diagnosis of FL was established by excisional biopsy of lymph nodes in 59 cases and by endoscopic biopsy of a colon polyp in one case. Using the World Health Organization classification system for histological grading, 20 cases were grade 1, 29 cases were grade 2, and 7 were grade 3. The remaining four patients had diffuse large B-cell lymphoma arising in association with an underlying FL. Three patients had discordant disease with large B-cell lymphoma of follicle center cell origin in the tissue biopsy specimens and low-grade FL in BM biopsy specimen. PB and BM aspirate samples represented initial pretreatment samples in 53 cases, and recurrent or residual disease in seven cases. The extent of FL involvement in BM was established by review of hematoxylin-eosin-stained sections of the decalcified, formalin-fixed trephine biopsy specimens. Based on the area involved by lymphoid aggregates and the overall BM cellularity, the percentage of BM involvement by FL was assessed semi-quantitatively, as 0 to 10%, 10 to 20%, etc. For the purpose of statistical analysis, the mid-point of the range (eg, 5%, 15%, etc) was used.

DNA Isolation and q-PCR
High molecular weight genomic DNA from PB and BM aspirate samples was extracted using the ABI 341 nucleic acid purification system (Applied Biosystems, Foster City, CA) or Autopure LS (Gentra Systems, Inc., Minneapolis, MN). Genomic DNA from frozen tissue was isolated using conventional phenol-extraction methods. The concentration of the DNA was determined by absorbance at 260 nm using a Beckman DU-7400 spectrophotometer (Beckman, Fullerton, CA).

All specimens including the control group were analyzed by TaqMan bcl-2/JH q-PCR coupled with fluorescent-based high-resolution capillary electrophoresis and GeneScan DNA fragment analysis (Applied Biosystems). The PCR reaction used 1 µg of genomic DNA and was described previously.23 All reactions were performed in duplicate. DNA obtained from the HL60 cell line was used as a negative control. Serial dilutions from 10–1 to 10–5-fold of a t(14;18)-positive follicular large cell lymphoma cell line,24 were used to generate standard curves. Amplification of a 93-bp sequence from the cyclophilin gene was performed simultaneously in all cases to normalize t(14;18) values to amount of input DNA.25 Following amplification, GeneScan analysis was conducted by capillary electrophoresis in an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). This method eliminates the need for gel electrophoresis and provides an accurate measurement of the size of the amplicon. By combining q-PCR with GeneScan analysis, we are able to eliminate the risk of error due to co-amplification of non-target DNA.23, 26

Statistical Analysis
Threshold cycle values of two replicates from each sample were averaged and the absolute bcl-2 mbr/JH and cyclophilin (Cy) levels were calculated by mapping the threshold cycle for each patient sample on a standard curve of known dilution of DNA from a positive control cell line. The bcl-2 mbr/JH to Cy ratio was then calculated to a normalized bcl-2/JH level. Because of the expected variance of PCR efficiency over different target concentrations, log10 (normalized bcl-2/JH) ratios were also computed to obtain a mean log difference between PB and BM aspirate samples.

We calculated correlation coefficients using a pair-wise comparison of bcl-2/JH levels in PB and BM aspirate samples. We similarly calculated correlation coefficients for bcl-2/JH levels in BM aspirate specimens with histological extent of FL involving the BM biopsy specimen. R-squared values were taken graphically and used to calculate correlation coefficients (r).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GeneScan analysis showed identically sized bcl-2 mbr/JH fusion sequences in paired PB and BM aspirate samples from all 60 patients with FL. The normalized PB and BM fusion levels were highly correlated in most patients regardless of whether or not there was histological evidence of FL in the concurrent BM biopsy specimen (Figure 1)Go . Following censure of the three most divergent samples because one sample of the pair appeared to have technical failure of the assay, a highly significant correlation coefficient (r = 0.887) was noted between the two sample types. The normalized bcl-2/JH level was higher in BM aspirate than in PB in 67% of cases and was higher in PB than BM aspirate in the remaining patients.



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  		Figure 1. Correlation between normalized bcl-2 mbr/JH levels in paired bone marrow aspirate and peripheral blood samples. A: Levels among patients in which concurrent BM core biopsy was involved by lymphoma. B: Levels among patients in which bone marrow biopsy was not histologically involved.

 
There was a highly significant correlation between the extent of FL seen in the BM biopsy specimen and the normalized bcl-2/JH level detected in concurrent BM aspirate samples (r = 0.71, Figure 2Go ). A less significant correlation was observed between PB bcl-2/JH level and extent of FL in the BM biopsy specimen (r = 0.55).



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  		Figure 2. Correlation between extent of follicular lymphoma present in bone marrow biopsy and level of normalized bcl-2 mbr/JH product in BM aspirate.

 
Among the 33 (55%) patients who had histological evidence of FL in the concurrent BM biopsy specimen, the median ratio of bcl-2/JH to cyclophilin was 8.01% in BM aspirate and 0.83% in PB samples. As shown in Figure 3AGo , 27 of 33 (82%) FL patients with histologically involved BM biopsy specimens had bcl-2/JH levels in PB and BM aspirate samples that were within one log difference; the mean log difference was 0.628. The levels of normalized bcl-2/JH in PB were not highly correlated with level of PB lymphocytosis (r = 0.362).



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  		Figure 3. Comparing levels of bcl-2/JH product in bone marrow aspirate and peripheral samples in the 60 patients with mbr+ follicular lymphoma. A: Comparison of BM and PB bcl-2/JH levels seen in patients who had lymphoma detected in concurrent BM core biopsy. B: Comparison of BM and PB bcl-2/JH levels in patients who did not have lymphoma detected in concurrent BM core biopsy.

 
Among the 27 (45%) FL patients who had no histological evidence of FL in the concurrent BM biopsy specimen, the median normalized levels of bcl-2/JH were 0.00615% in BM aspirate and 0.0095% in PB samples, with a high correlation between the values from the two sample types (r = 0.93). Twenty-five (93%) of these patients had normalized bcl-2/JH levels in BM and PB that were within one log difference (Figure 3B)Go . The mean log difference between PB and BM aspirate levels in this group was 0.35. Twenty-five of 27 (59.3%) patients had higher normalized levels in PB than in BM aspirate. Sixteen FL patients had normalized bcl-2/JH levels that were 0.015% or less, representing 59% of FL patients with negative BM biopsy specimens and 27% of all FL patients studied. In the three patients with the lowest normalized bcl-2/JH levels, PCR and GeneScan analysis of the primary tumor revealed a bcl-2/JH fusion sequence of identical size to that detected in the PB and BM aspirate samples. These results demonstrate that the low levels of bcl-2/JH detected in PB and BM aspirate samples of these three patients were derived from the neoplastic clone.

For comparison, we studied 15 paired PB and BM aspirate samples from patients with B-cell lymphoproliferative disorders other than FL who also had low levels of bcl-2 mbr/JH detected. In these patients, the level of bcl-2/JH ranged from 0.0002 to 0.014 in PB and 0.0001 to 0.012 in BM aspirate samples (Table 1)Go . In three cases, the bcl-2/JH fusion sequences were detected only in the BM or the PB samples. The diagnoses for these 15 cases were chronic lymphocytic leukemia (n = 7), extranodal marginal zone B-cell lymphoma (n = 4), anaplastic large cell lymphoma (n = 1), marginal zone B-cell lymphoma (n = 1), mantle cell lymphoma (n = 1), and acute lymphoblastic leukemia (n = 1). Because of the low levels of product and the absence of FL based on the results of tissue biopsy and flow cytometric characterization of the PB and BM samples, we attribute these bcl-2/JH fusion products to background non-neoplastic or preneoplastic lymphocytes carrying an occult t(14;18).


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  		Table 1. Comparison of bcl-2 mbr/JH Levels Detected in PB and BM Samples

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was designed to address whether levels of bcl-2 mbr/JH fusion sequence detected by q-PCR in PB and BM aspirate samples obtained from FL patients are similar, thereby obviating the need for BM examination solely for this purpose. We included 60 consecutive FL patients with the t(14;18) involving mbr who had q-PCR analysis performed on paired PB and BM aspirate samples. Overall, there was a strong correlation between levels detected in PB and BM aspirate samples, however, because we had a limited number of cases with extensive BM involvement, the absolute correlation between BM aspirate and PB samples may be somewhat overstated. In cases with moderate to high levels of bcl-2/JH, both PB and BM aspirate samples usually had high levels of product but in 33% of cases, the normalized bcl-2/JH levels were higher in PB than in parallel BM aspirate samples. These results closely approximate those reported previously by Summers and colleagues21 and suggest that PB can be used in place of BM aspirate samples for determination of translocation status.

Samples from 16 (27%) FL patients in this study had low bcl-2 mbr/JH levels detected in PB and BM aspirate samples that were in the range that was occasionally seen in patients without FL, namely 0.015% (bcl-2/JH divided by cyclophilin) or less. Assuming equal efficiency of amplification for bcl-2 mbr/JH and cyclophilin DNA targets, this ratio approximates the number of cells bearing a t(14;18) compared to the overall number of cells analyzed. Thus, there is a threshold of approximately 1 bcl-2 mbr/JH-bearing cell in 3000 cells that defines a lower limit below which the fusion sequence detected could be derived from occult translocations in non-neoplastic or preneoplastic cells (ie, "background levels").8, 9, 10, 11, 12 In this circumstance, concurrent analysis of the primary tumor to confirm the true translocation status is likely required for definitive interpretation of the significance of a low/borderline bcl-2/JH level in PB or BM aspirate samples.12

This analysis is supported by previous work by Tsimberidou et al22 who reported low/background levels of bcl-2 mbr/JH fusion sequences in PB specimens obtained from 22% of non-FL donors, with a frequency as high as 40% when a 10-fold excess of genomic DNA (10 µg) was used for amplification. They also reported that 2 to 3% of non-FL samples had bcl-2/JH levels that overlapped with levels that can be detected in FL samples. Previous research indicates that t(14;18) translocations are spontaneously generated in lymphocytes of healthy individuals, and that such cells only rarely progress to overt lymphoma.9, 12 Furthermore, age appears to have a significant influence on the likelihood of translocation detection, with older individuals more frequently having positive PB samples, and also having a much higher fusion product levels.8

The correlation between bcl-2/JH levels detected in BM aspirate samples and the approximate extent of FL involving the concurrent BM biopsy specimen also suggest that q-PCR may be used as an alternate method for determining the presence and general extent of BM involvement by FL. As expected, the concordance between morphological findings and q-PCR was less significant at low bcl-2/JH values where histological involvement may be difficult to detect. Although there was a trend toward a correlation, bcl-2/JH levels in PB samples were less useful for predicting the extent of lymphoma involvement in BM biopsy specimens, indicating that the absence of bcl-2/JH positivity by PCR in PB samples cannot be used to absolutely predict the absence of BM involvement by FL.

At our institution, patients with FL commonly are first diagnosed by tissue biopsy elsewhere, and fresh tissue or paraffin blocks of the primary tumor are often not available for molecular testing. In these cases, in terms of patient comfort and expense, a PB sample is clearly preferred over BM aspiration for determination of translocation status. We believe this study demonstrates that real-time q-PCR combined with capillary electrophoresis and amplicon sizing using GeneScan analysis can reliably allow analysis of bcl-2 mbr/JH status in PB samples in lieu of BM aspiration. This approach is highly sensitive when compared with non-quantitative PCR. By accurate size comparison of the fusion sequences between two sites, GeneScan analysis also allows one to eliminate false-positive results due to non-lymphoma-associated translocations.23, 26 Furthermore, the correlation between the results obtained from PB and BM aspirate samples at all levels of disease (ranging from very high to very low levels of bcl-2/JH), suggests that PB analysis is likely to be as informative as BM aspirate analysis for monitoring tumor burden in FL patients over time.

However, for newly diagnosed cases of FL with low bcl-2/JH levels in PB or BM aspirate samples, confirmation of the bcl-2/JH amplicon size by testing of the primary tumor is justified. The data in this study also suggest that high levels of bcl-2/JH detected in PB or BM aspirate samples can be used as a substitute for testing of the primary neoplasm. However, as already stated, relatively low levels of bcl-2/JH detected in PB and BM aspirate samples may not be derived from neoplastic cells, necessitating a repeat analysis on a tissue biopsy, or obtaining the original biopsy specimen, if available, for bcl-2/JH analysis.


   Footnotes
 
Address reprint requests to Rajyalakshmi Luthra, Ph.D., The University of Texas M.D. Anderson Cancer Center, NA01.091, 8515 Fannin Street, Houston, TX 77054. E-mail: rluthra{at}mdanderson.org

Accepted for publication June 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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