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Immunoglobulin heavy chain gene rearrangements assays for improvement of clinical diagnosis of Hodgkin Lymphoma.


Hodgkin's lymphoma (HL) is rare of the lymphoma malignancy, which is originates from B -lymphocyte cells [1]. According to immunopathological features and histomorphology, HL is also categorized in to the classical Hodgkin's lymphoma (cHL) and nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL) [2, 3]. Importantly, HL is an uncommon subtype of lymphoid disorder in good economic country, whereas it is the most frequent malignancies in the west geographic territory [4]. Classical HL comprises several sub-types such as, nodular sclerosis (NS), mixed cellularity (MC), lymphocyte depletion (LD), and lymphocyte-rich (LR) Hodgkin lymphoma [5]. Cancerous cells in cHL are known as Hodgkin and Reed-Sternberg (HRS), which originate from germinal center (GC) or post-GC in B cells and carried somatic hypermutations in variable genes of IGH [6]. Due to scarcity HRS cells population, failure of the surface antigen expression, and acquired somatic mutation, cancerous cells lineage determination by immunophenotyping and molecular approaches is very difficultly [7, 8].

Amplification of Ig genes rearrangements have been proposed as a appropriate method to lymphoma diagnosis (9). Currently, few investigations were performed to clonality detection in HL cases (10, 11-13). In the present study, we applied BIOMED-2 multiplex PCR kit, to evaluate gene rearrangements clonality in IGH for improvement of diagnosis and for increasing sensitivity of pathological features of HL. In the PCR when one band in PCR product background was detected, monoclonality was confirmed. Observation of more than four clonal rearrangements was considered oligoclonality and multiple rearrangements clone were interpreted as polyclonality.


Samples preparation:

This investigation was performed on 50 Formalin Fixed, Paraffin-Embedded (FFPE) specimens diagnosed with HL by immunophenotyping and morphological assessment. All of the FFPE blocks were recruited between 2011-2013. Criteria for samples selection was definite diagnosis of HL. The subjects whose specimens were studied had no history of the others hematologic malignancies as re-evaluated by pathological board on the bases of histological features and immunohistochemical (IHC) staining.

DNA Extraction:

To evaluate molecular clonality, three slices of 10p FFPE tissue were used for extraction of DNA according to the protocol previously described by Maria et al [13]. To assessment quality and quantity extracted of DNA, amplified BIOMED-2 control genes and UV spectrophotometry (260/280 nm using the NanoDropTM ND1000, NanoDrop Technology, Wilmington, DE, USA) [18]. The mean DNA concentration was 400ng/pl and OD260/280 ratio was 1.85. All of the samples were interpreted according to the Euro/Clonality BIOMED-2 guideline [14].

Clonality analysis:

We used a gold standard multiplex PCR protocol provided by European Biomedicine and Health (BIOMED-2) Concerted Action Project BMH4-CT98-3936 for improvement of diagnosis and analysis of clonality gene rearrangement in lymphoma malignancies.

Following amplification, PCR products were denatured for 5 minutes at 94 C, were then incubated for 60 minutes at 4 C. 10 pl of products was loaded on non-denaturing polyacrylamide gel (8%) and stained with silver nitrate. In each run of electrophoresis, lymphatic tissue (Tonsil) was used as a polyclonal DNA sample, sterile water as negative and the sensitivity panel IVS-0010 (5%) control clonal DNA (InvivoScribe; catalog No. 4-088- 0590) as positive sample. Clonality analysis was performed using amplification on three frame woke of IGH (VH FR1-Jh, Vh FR2-Jh, and VH FR3-JH) gene rearrangements using the multiplex PCR protocols suggested by BIOMED-2 [14]. To increase accuracy of detected clonality, all samples were assessed in duplicate.

Statistical Analysis:

To determinate, relationship between variations of CD30 marker expressed in cancerous cells and clonality rates, the Pearson's chi-square ([chi square] test) and Fisher's exact tests were performed. The results obtained were analysed using SPSS statistical software, version 19.0 (SPSS Inc., Chicago, IL, USA). The P-values < 0.05 were considered as significant statistically.


Molecular Gene Rearrangement Analysis:

Of the 50 FFPE tissue blocks evaluated, 43 (86%) were cHL and 7 (14%) were NLPHL. CHL consisted of 8 (19%) cases with nodular sclerosis (NS), 10 (23%) cases with mixed cellularity (MC), one (2%) case with lymphocyte depletion (LD) and 24 (56%) of cases cHL sub-types of the which were not determined to. Following amplification, a total of 74% (37/50) positive monoclonality rearrangements were detected in IGH gene. Thirteen of cases (13/50, 26%) not showed any monoclonality. The most frequent gene rearrangements were detected in IGH assay related to FRIII (50%) and FRII (24%). Conversely, in FRI analysis, no clonality was identified in any of the samples (Table 1). Statistically significant association between positive CD30 markers expressed in cancerous cells and monoclonality gene rearrangements in immunoglobulin genes was observed. The percentages of CD30 expression using IHC staining in HRS cells were showed in Figure 1.

Regarding number of positive CD30 cells and IGH monoclonality, statistically significant relationship was found (p < 0. 05).


The purpose of this investigation was design evaluation of an auxiliary approach to increase sensitivity of diagnosis and to apply BIOMED-2 molecular gene rearrangements assays on FFPE tissue in HL samples. We studied clonality rearrangements in IGH gene in 50 FFPE cases diagnosed as several sub-types of HL consisting to NS, MC, LD, and NLPHL neoplasms. We analysed three FRI, FRII, and FRIII of complete IGH rearrangements. Monoclonality rearrangements were identified in 37 out of 50 (74%) HL cases. Regarding rate of clonal rearrangements in FRI-JH, no monoclonality was detected in any of the sub-types of HL. Our findings are compatible with previous investigations [7, 9-12]. In analysis of FRII-JH and FRIII-JH rearrangements, we demonstrated positive clonality in 71% (31/43) and 86% (6/7) of cHL and NLPHL types, respectively. The 74% sensitivity of clonality detection reported here, is a significant improvement over with to other investigations has previously been reported 15.5%, 44.5%, 50%, 10%, and 24% [7, 9-12]. In the present study, we have shown 86% monoclonality in IGH assay within NLPHL cases, whereas others [12, 17] no reported any positive clonality. Several factors such as histological structures, sample types, and specificity assessments have been reported to have significant effect on sensitivity of monoclonality rearrangements assay applying BIOMED-2 protocols [14]. We also, assessed the relationship between positive CD30 cells and rate of IGH clonality rearrangements. In confirmation of previous reports and other investigations [9, 11, 12], we found (p = 0.000) increases as the number of positive CD30 cells, the rate of monoclonality rearrangements assessed will be enhanced. Thus, it is conceivable that one of the most differences positive clonality was detected depended to the density of cancerous cells. However, further studies are needed to clarify these differences. Although, investigators have applied different methods (Gene Scanning and Heteroduplex gel electrophoresis) to detect rearrangements, both approaches have similar sensitivities in clonality PCR assays [14]. In addition, the relatively high percentage of clonality in our study in comparison to previous investigations may be due to the relatively greater conditions of the DNA samples and high density of neoplastic CD30 positive cells.


Overall, detection rate of clonal gene rearrangements in HL is less than NHL. In addition, monoclonality detection rate is closely related to the density of cancerous cells (HRS). We conclude that analysis of clonal gene rearrangements in IGH using BIOMED-2 protocols could be implemented as a valuable and reliable method to increase sensitivity and accuracy of determining HL similarly to NHL. However, in clinicopathological diagnosis of neoplasms, the sensitivity of the applied methods that should be 100%, Therefore, in addition analysis of IGH gene rearrangements, IGK, IGH D-J, and IGL clonality assessments seems to be required for optimal sensitivity diagnosis.


Article history:

Received 11 June 2014

Received in revised form 21 August 2014

Accepted 25 September 2014

Available online 25 November 2014


This study was supported by the Iran National Science Foundation (INSF, Grant No: 87041113). We gratefully acknowledge Dr Dastmalchi and Dr Hamzeh-Mivehroud from the Biotechnology Research Center, Tabriz University of Medical Sciences for assistance during this study.

Declaration of Interest:

The authors declare that there is no conflict of interest.


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(1) Saeid Ghorbian, (2) Golchin Moharrami, (3) Issa Jahanzad, (4) Mehrdad Asghari Estiar, (5) Iraj AsvadiKermani, (5) Jamal Eivazi Ziae, (1) Gholam Reza Javadi, (6) Ebrahim Sakhinia

(1) Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

(2) Department of Medical Genetics, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

(3) Department of Pathology, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran

(4) Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tabriz, Iran

(5) Hematology and Oncology Research Center, Tabriz University of Medical Science, Tabriz, Iran

(6) Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Corresponding Author: Gholam Reza Javadi, Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Table 1: The combination of three FR (I, II, and III) IGH clonality
gene rearrangements assessed according to BIOME-2 protocols. NLPHL,
Nodular lymphocyte-predominant Hodgkin's lymphoma; cHL, Classical
Hodgkin's lymphoma. NS, Nodular Sclerosis; MC, Mixed Cellularity; LR,
lymphocyte-rich; LD, Lymphocytic depletion.

Diagnosis                            IGH

                        [FR.sub.I]   [FR.sub.II]   [FR.sub.III]

cHL          NS             0             0            5/8
             n=8           (0%)         (0%)         (62.5%)
             MC             0           2/10           6/10
            n=10           (0%)         (20%)         (60%)
             LD             0            1/1            0
             n=1           (0%)        (100%)          (0%)
        HL (No-typed)       0           8/24           9/24
            n=24           (0%)        (33.5%)       (37.5%)
NLPHL        LR             0            1/7           5/7
             n=7           (0%)        (14.3%)       (71.5%)
            Total           0           12/50         25/50
            n=50           (0%)         (24%)         (50%)

Diagnosis                             IGH

                        [FR.sub.I] +   [FR.sub.I] +
                        [FR.sub.II]    [FR.sub.III]

cHL          NS              0             5/8
             n=8            (0%)         (62.5%)
             MC             2/10           6/10
            n=10           (20%)          (60%)
             LD             1/1            1/1
             n=1           (100%)         (100%)
        HL (No-typed)       8/24           9/24
            n=24          (33.5%)        (37.5%)
NLPHL        LR             1/7            5/7
             n=7          (14.3%)        (71.5%)
            Total          12/50          25/50
            n=50           (24%)          (50%)

Diagnosis                             IGH

                        [FR.sub.II] +   [FR.sub.I] +
                        [FR.sub.III]    [FR.sub.II] +

cHL          NS              5/8             5/8
             n=8           (62.5%)         (62.5%)
             MC             8/10            8/10
            n=10            (80%)           (80%)
             LD              1/1             1/1
             n=1           (100%)          (100%)
        HL (No-typed)       17/24           17/24
            n=24            (71%)           (71%)
NLPHL        LR              6/7             6/7
             n=7            (86%)           (86%)
            Total           37/50           37/50
            n=50            (74%)           (74%)

Fig. 1: Immunophenotype features of the surface antigen receptors
expressed (CD30) in cancerous cells (HRS cells) in HL by
immunohistochemical staining. NS, Nodular Sclerosis; MC, Mixed
Cellularity; LR, lymphocyte-rich; LD, Lymphocytic depletion.

CD30 Marker                 Number of Case

                    CD30/Positive   CD30/Negative

MC                       90%             10%
NS                       75%             25%
LD                       100%             0%
HL-Non Classified        85.5%           12.5%
LR                       57.2%           42.8%

Note: Table made from bar graph.
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Article Details
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Author:Ghorbian, Saeid; Moharrami, Golchin; Jahanzad, Issa; Estiar, Mehrdad Asghari; AsvadiKermani, Iraj; Z
Publication:Advances in Environmental Biology
Article Type:Report
Date:Nov 15, 2014
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