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Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 903-908

Novel diagnostic biomarker for patients with Non-Hodgkin's Lymphoma by IgH gene rearrangement

1 Department of Hematology, Chinese PLA (People's Liberation Army) General Hospital, Beijing 100853, China
2 Departments of Pathology, Chinese PLA (People's Liberation Army) General Hospital, Beijing 100853, China
3 Department of Hematology, Tangdu Hospital, Fourth Military Medical University, Xian 710038, China

Date of Web Publication25-Jul-2016

Correspondence Address:
Honghua Li
Department of Hematology, Chinese PLA (People's Liberation Army) General Hospital, Beijing-100 853
Yu Zhao
Department of Hematology, Chinese PLA (People's Liberation Army) General Hospital, Beijing-100 853
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.157345

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 > Abstract 

Aim of Study: Novel biomarkers for improving accuracy could be beneficial for disease monitoring and surveillance of Non-Hodgkin's lymphomas (NHL). So we explored the viability of analytical methods for identifying the rearranged immunoglobulin (Ig) H genes sequence.
Materials and Methods: Next-generation sequencing (NGS) was used to sequence deoxyribonucleic acid (DNA) extracted directly from the tumor tissues of patients with NHL, and then specific rearranged DNA fragments from plasma was detected by polymerase chain reaction (PCR).
Results: By parallel DNA capturing and sequencing of IgH genomic regions (IgCap), the sequence of rearranged IgH loci could be detected and precisely determined in tumor tissues of 12 patients with NHL. The circulating rearranged DNA fragments had been identified in the plasma of one patient.
Conclusion: IgCap may be the favorable diagnostic method for patients with NHL in clinical.

Keywords: Biomarker, gene rearrangement, Non-Hodgkin's lymphoma

How to cite this article:
Bo J, Sun L, Wang W, Ma C, Li H, Zhao Y. Novel diagnostic biomarker for patients with Non-Hodgkin's Lymphoma by IgH gene rearrangement. J Can Res Ther 2016;12:903-8

How to cite this URL:
Bo J, Sun L, Wang W, Ma C, Li H, Zhao Y. Novel diagnostic biomarker for patients with Non-Hodgkin's Lymphoma by IgH gene rearrangement. J Can Res Ther [serial online] 2016 [cited 2020 Jul 12];12:903-8. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/903/157345

Jian Bo and Lu Sun contribute equally to this work.

 > Introduction Top

Non-Hodgkin's lymphoma (NHL) is a type of cancer that originates in the lymphatic system. It is estimated that lymphoma is the sixth most common cancer in the United States.[1],[2] NHL is diagnosed by a tissue biopsy. If there is an enlarged, painless lymph node, without of an infection, a biopsy will be needed. For an aggressive type of lymphoma, a combination of chemotherapy and biological therapy is usually indicated, and sometimes radiation therapy will be added, although close follow-up is necessary.

For improving cancer chemotherapeutics, it is necessary to monitor therapy effects which is more important.[3] The good marker should be one that could be simply assessed without exposure to irradiation and repeat biopsies, is totally precise for carcinoma (to avoid false positives) and susceptible for disease with cost-effective.[4] In the lots of new developing biomarkers, the somatically deoxyribonucleic acid (DNA) sequences with mutation in the plasma are circulating and particularly attractive.[5],[6] Moreover, mutations are gracefully tumor-specific that could not be found in normal cells come from patient, and it offers many advantages over other markers associated with carcinomas, such as prostate-specific antigen (PSA) or carcinoembryonic antigen (CEA).[7] The mutant DNA present in circulation could be found in various solid tumors, furthermore, they provide specificity and sensitivity indicators superior to conventional disease through initial studies.[8] Leukemias belong to liquid tumors; the fused genes (like BCRABL) consistently provide extremely helpful markers for patients.[9] In patients with leukemia, the rearrangement mutated genes could be used for accessing effect of therapy according to the fact that any residual cancer cells exist in bone marrow or blood.[10] But, circulating cells are not found in blood or marrow of patients with tumors such as lymphomas. The templates of somatically rearranged DNA that come from patients with lymphomas could be found in blood or plasma as the cell-free fraction.

To explore an effective tool, it was primarily necessary for assistant diagnosing NHL to identify abnormal DNA sequences in patients with lymphoma. Though no explicit site mutations or rearrangements in oncogene has been found in patients with lymphomas; the immunoglobulin (Ig) genes rearrangements exist in all lymphomas. These rearrangements have been described with intelligent assays, and was mainly using multiplex polymerase chain reaction (PCR) to clone rearrangements by electrophoresis, the increased warning sign of a piece 'spike' represented the rearrangement.[11] In many situations, such examines are clinically useful, but it is difficult to detect the clonal rearrangements 'spikes', moreover due to the primers that could not anneal to the hypermutated rearranged Ig genes sequences. More importantly, such examines are not most favorable sensitivity, because these tumor-specific rearrangements are overlaid on the normal Ig rearrangements. Therefore, these tumor-specific rearrangements could only be found if their abundance is considerably larger than the summative level of rearrangements come from normal B cells. However, the form of such rearrangements in plasma associate with the complicated lymphoma and perseverance of such rearrangements next chemotherapy, it seems to indicate fine prognosis for NHL.[12] To improve the specific plasma-based approach at the sequence level, the more specific capture-and-sequence method was utilized to identify rearranged IgH genes then to distinguish the same rearrangements in plasma of patients with NHL.

 > Materials and Methods Top

Patients and samples

The current study includes 16 patients diagnosed with NHL based on histopathologically and immunohistochemically in our hospital from February 2010 to July 2014. It included 12 cases (75%) of diffuse large B-cell lymphoma (DLBCL); four cases (25%) were follicular lymphoma (FL). Biopsy pathology diagnoses were according to the 2008 World Health Organization (WHO) classification of lymphomas.[13]

All studies were approved by the Ethics Committee of Chinese Institution and each patient had given informed consent. The use of case data in this research was approved by the ethics committee in our hospital according to the Declaration of Helsinki. We clearly confirm that we had all the necessary consent from any patient involved in the study, including consent to participate in the study and consent to publish.

DNA purification

DNA was taken out from 1,000 μL plasma with the QIAamp Circulating Nucleic Acid kit (QIAGEN; Valencia, CA) following the instructions of manufacturer. DNA sample was taken out from 10 slides of formalin-fixed paraffin-embedded (FFPE) tissue samples following Qiagen AllPrep kit manufacturer's protocol, and quantified by NanoDrop 2000. DNA library was prepared using Illumina library preparation kit with at least 100 ng initial DNA according to the provider's protocol. In order to get enough DNA libraries, the final PCR step was increased to 15 cycles.

IgCap capture

The target genomic region was captured using VDJ enrichment kit (MyGenostics Inc., Beijing, China). The targeted capture region included all V-, J-, and D-gene exons. The target enrichment is conduced according to manufacturer's protocol. In brief, 1 μg DNA library was mixed with Buffer BL and GenCap VDJ Probe (MyGenostics, Beijing, China), heated at 95°C for 7 min and 65°C for 2 min on a PCR machine; 23 μl of the 65°C prewarmed Buffer HY (MyGenostics, Beijing, China) was then added to the mix, and the mixture was hold at 65°C with PCR lid heat on for 22 h for hybridization. Fifty microliters MyOne beads (Life Technology, Carlsbad, CA) was washed in 500 μL × 1 binding buffer for three times and resuspended in 80 μl × 1 binding buffer. Sixty-four microliter ×2 binding buffers was added to the hybrid mix, and transferred to the tube with 80 μl MyOne beads. The mix was rotated for 1 h on a rotator. The beads were then washed with WB1 buffer at room temperature for 15 min once and WB3 buffer at 65°C for 15 min three times. The bound DNA was then eluted with buffer elute. The eluted DNA was finally amplified for 15 cycles using the following program: 98°C for 30 s (one cycle); 98°C for 25 s, 65°C for 30 s, and 72°C for 30 s (15 cycles); and 72°C for 5 min (1 cycle). The PCR product was purified using SPRI beads (Beckman Coulter) according to manufacturer's protocol. The enrichment libraries were sequenced on Illumina HiSeq 2000 sequencer for paired read 100 bp.

Bioinformatics analysis

After HiSeq 2000 sequencing, high-quality reads were retrieved from raw reads by filtering out the low quality reads and adaptor sequences using the Solexa QA package and the cutadapt program (http://code.google.com/p/cutadapt/), respectively. The first 35 bases from read 1 and last 35 bases from read 2 were separately trimmed from the reads. These short sequences were matched to the human genome, allowing at most two mismatches. If the distance of the two matched positions was longer than 2,000 bp, the sequencing reads were retained, otherwise, filtered. The remaining reads were considered to be VDJ recombination sequences if both the two short sequences fell into the VDJ regions. The selected paired reads were then assembled to a single sequence according to the overlapping bases. The colonel VDJ recombination was defined by more than three distinct sequences covering the same recombination junction.

 > Results Top


To identify the rearranged DNA sequences instead of ribonucleic acid (RNA) since adequate quality of RNA is not accessible to a lot of clinical situations, but DNA is able to obtain willingly from samples. IgCap engages three actions: (a) At first, DNA of tumor is cut off and ligated to adapters at random that make amplification of their subsequent by PCR available; (b) IgH fragments are captured based on the hard sustain containing the interesting sequences; [12],[14] (c) by traditional PCR, captured DNA is amplified and generate the library of IgCap, the terminators of these captured DNA fragments are focused on extremely parallel sequencing.[12]

Then obtained sequences were processed to identify rearranged sequences in silico. All captured IgCap fragments contained applicable sequences of IgH gene, but not only the rearranged DNA fragments. The rearranged sequences were almost constantly mutated, both at the margins of the rearranged exons and in the exons. Both these characteristics identify the challenge of rearrangements, especially when merely a quite small amount of bases are determined from each fragment.

The rearranged IgH genes could be ascertained based on numerous features and discerned from unrearranged genes according to develop algorithms. The original algorithm (called CTGGGG-algorithm) ascertained 'seed' fragments that present in all J genes (CTGGGG) from a conserved sequence, included the six nucleotide sequence which was same as, or differed at one site. The next algorithm (paired-end algorithm), utilized paired-end reads to ascertain two series of seed fragments. The first series fragment was related to normal V, D, or J regions; however whose terminates signified sequences isolated by >10,000 bp in the expected orientation and in unrearranged DNA. The next series contained fragments in which one of the two terminates was associated to the conventional V regions and the other terminate contained specific sequences inside J or D regions.

The specific J region sequence was CTGGGCCA, while the specific D region sequences contained five bases in the middle of each D regions. These seed fragments were broadened to contain larger regions of V, D, or J in both algorithms by achieving searches about homology in the sequenced IgCap library with the other fragments.[12]

Detection of rearranged DNA of IgH genes in tumors

Although the above results illustrated that we could ultimately ascertain rearrangements in 12 of the 16 patients [Table 1] and [Table 2]. The quantity of IgCap library fragments including sequences matching rearranged gene was commonly miniature in patients with a rearranged identified fragment from tumor tissues. DNA in plasma is surely not augmented in rearranged fragments, and nearly all IgH gene contains fragments of DNA in plasma which are indisputably original from nontumor tissue. In patient 7, IgCap identified two rearrangements that were represented [Table 2]. In the patient, one of the two rearrangements was probably derivative from paternal allele and the other from maternal allele.
Table 1: Characteristics of patients identified rearrangements in this study

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Table 2: Rearrangements identified in this study

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Notably, the number of fragments containing sequences in the IgCap library linked the rearrangement was significantly more than one in every case. In these rearrangements, we could recognize VD or VDJ junctions. It could not define whether it was due to technical or biologic reasons, and is well-known that partial rearrangements happen in neoplastic B-cells, in which V is not fused to D but D is fused to J.[15]

Recognizing rearranged DNA of IgH genes from plasma

According to the method described above, we are going to straightly recognize rearranged DNA in IgH gene from patient's plasma. We applied the same procedure from the plasma of one health control without B-cell neoplasia, and did not found any rearrangements. Then we performed DNA amplification from the above 12 patients with NHL base on their identified rearrangements by PCR from corresponding plasma samples. Only for one case (patient 2), PCR products with estimated dimension in his plasma was obtained, but on the contrary, none was identified from control group [Figure 1]. The amplified fragments by PCR were removed from the sequence, and then cloned. In this situation, the sequence of this patient was foreseen from the algorithm (could have amplified from clone, whole genome, or clonal progression excluding a single base substitution). After radiotherapy, the result of her was negative from plasma. In the patient, rearranged fragments were obviously and could be examined in very little plasma (30 ml). These results are uniform with the theory, circulation lymphoma cells are so rare that the DNA with rearrangements liberated from tumors in situ was much more than originated from lymphoma cells in circulating.
Figure 1: PCR amplification of Ig rearrangements in tumors and plasma. The Ig rearrangement was shown for sample of patient 2. DNA was purified from the plasma of the patient (1–5#) or from normal cells of an unrelated patient (control: 6# and 7#). Marker, molecular weight markers, and size indicated on right in base pairs (bp). PCR = Polymerase chain reaction, Ig = Immunoglobulin, DNA = Deoxyribonucleic acid

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 > Discussion Top

The main target is to identify new molecular markers that may be used in rapid and economic tests, which should be not invasive for patients with NHL. The easier diagnostic strategies, which would allow stratifying for biopsy for only patients at risk should be investigated.

The results illustrated above showed that rearrangements of IgH genes can be regularly identified in tumors of patients with NHL, and are present at measurable levels in circulation. Moreover, the circulation DNA is more plentiful than lymphoma cells in circulation. He et al.,[12] found that if no biopsy for tumor is accessible, IgCap method is dominant enough to identify rearrangements of IgH genes in some patients straightly from plasma. We found rearranged gene from plasma of one case only which might relapse. After radiotherapy, the result of her was negative in plasma. We thought that they reached long-term complete remission after treatment, then rearranged gene could not be found from their samples. These outcomes settle on the stage for clinical achievement by this biomarker to follow patients with NHL next therapy.

The assessment of rearrangements in T-cell receptor genes or Ig by clone method has a long history.[11] At first, the gold standard for this kind of analysis is southern blotting. However, it required large quantity of genomic DNA with high quality, and the diagnostic test sensitivity was low. Because of many various advantages, PCR-based assays substituted for southern blotting. The multiplex PCR assays facilitated by designing primer sets rigorously, which was identified up to 96% FL and 74% DLBCL.[16] Massively parallel sequencing of PCR products obtained from samples according to the above method has lately been used to confirm VDJ rearrangements in T-cell receptor or Ig genes.[17],[18],[19] Herein, VDJ genes are captured by using equivalent sequence method from large amplification products, and specific primers for Ig genes. It indicated that one more step towards the recognition of clonal rearrangements might be used as biomarkers. Even if more complicated than the direct approaches by PCR, the advantage of the biomarker was low bias. In fact, it could distinguish 100% of rearranged fragments from tissues of patients with lymphoma. In addition, it can be used to degrade DNA samples that originated from those paraffin-embedded tissues or plasma. The degraded DNA was not amplified efficiently with commonly used primer sets, because the amount of amplified product was bigger than its DNA. The two algorithms of He et al.,[12] was utilized to ascertain rearranged Ig genes. We used the algorithm based on CTGGGG sequence and it could determine all classic rearrangements of Ig genes. However, this algorithm was not able to determine rare rearrangements of Ig genes, for example, Ig rearrangements only involving V and D region or with deletions in J region. The algorithm of paired-end is a supplement.

Here, the method illustrated was limited, such as all biomarkers. The main theoretical limitation was stability of the marker. Undergoing clonal divergence so that the rearrangement of relapsed lymphoma may be immeasurable with the primers utilized to confirm the original tumor by PCR.[20] Hence, in our study, it was so comforting that the tumor samples of patient 7 with NHL could be evaluated, and two distinct rearrangements was obtained from her samples. During follow-up, the two individual markers was so availability that diminish the chance of false negative diagnose. With our method, the sequence of rearrangement is exactly detected. By two sets of primers, every rearrangement could be explored, thus it could reduce the latent influence from clonal evolution on site of rearrangement in Ig genes.

The method or medicine for treatment of DLBCL has been developed with huge enthusiasm, especially in risk-adapted therapy. For guiding judgments on switching chemotherapy regimens and selecting high-dose chemotherapy as consolidation treatment, fluorodeoxygenase-positron emission tomography (FDG-PET) was utilized.[21],[22] Nevertheless, standardized analysis of PET imaging even showed barriers, as lately noted in a cooperative group clinical experiment.[23] Herein, the plasma-based method illustrated stands for an alternative, and it may be the supplement approach to assessing tumor treatment response. In the future, another vital matter to be noted is the sensitivity of the approach. The limitation of our study was that patients were assessed only after treatment. During therapy, it is too difficult to determine rearrangements of IgH genes in plasma of patients with residual minimal disease. Rearrangements can be detected in as little as 30 ml of plasma in patient 2 whose levels were quantified. It demonstrated that amounting to only 1% of disease burden in patients participated in our research can be easily determined in 3 ml plasma, if presuming the relationship between plasma DNA and disease burden is linear. After radiotherapy, the result of the patient from plasma was negative. So we thought that the rearrangements detected with IgCap may be used to define recurrence. As illustrated above, this biomarker could be comforting supplied to plasma by PCR. Though it is available, but it is obviously favorable to confirm the rearranged biomarker by using the IgCap strategy on DNA come from patients with lymphomas, better than from plasma.

In conclusion, IgCap is an personalized and advanced approach for treatment on patients with NHL, which supplies exciting break in research and provides the probability for supplemental method in clinical diagnosis on patients with NHL. The essential thought of IgCap may be supplied to diagnose and assess NHL, to identify the amount and nature of rearrangements Ig genes in samples that come from patients.

 > Acknowledgement Top

This work was supported by grants from the National Natural Science Foundation of China (No. 30600524, No. 81201758 and No. 81071990) and the Foundation of Translational Research in Chinese PLA (People's Liberation Army) General Hospital (2013FC-ZHCG-1001). The study sponsors had no involvement in the study.

 > References Top

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  [Figure 1]

  [Table 1], [Table 2]


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