Detection Of Immunoglobulin Gene Rearrangements Research Articles

Detection of Second Primary Lymphoma in Late Diffuse Large B-cell Lymphoma Recurrences

Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing–based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein–Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing–based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies.

Clonality assessment and detection of clonal diversity in classic Hodgkin lymphoma by next-generation sequencing of immunoglobulin gene rearrangements

AbstractClonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.

Solitary plasmacytoma of bone: a clinicopathologic, immunohistochemical and immunoglobulin gene rearrangement study

To investigate clinicopathologic features of solitary plasmacytoma of bone (SPB) and the role of immuno-phenotype and immunoglobulin gene rearrangement detection in the diagnosis and differential diagnosis of SPB. A total of 21 cases of SPB were selected during a period from 1990 to 2008. A retrospective clinicopathologic study and immunohistochemistry (EnVision or EliVision methods) of 17 antigens were performed. In addition, universal IgH (FR3A/LJH/VLJH) primers and BIOMED-2 PCR multiplex tubes were used for IgK and IgL rearrangement analysis. The age of patients ranged from 36 to 72 years with a media of 50 years. Axial skeleton was the most common site of involvement, accounting for 66.7% of the cases (14 of 21), followed by the extremities of 33.3% (7 cases). Low serum level of M-components was found in 5 cases, including two of IgG type (21.4 g/L) and three of IgA type. Clinical manifestations were closely related to the anatomic sites involved, such as pain due to bone destruction, symptoms and signs caused by compression of spinal cord or nerve root, and pathological fracture. All cases presented as a solitary osteolytic lesion. According to the histological grading criteria, grade I tumor was seen in 12 of 21 cases (57.1%). The remaining were grade II (5 cases, 23.8%) and grade III (4 cases, 19.0%). Immunohistochemically, the neoplastic cells expressed two or more plasma cell antigens, including CD138, CD38 and PC, but no CD19 and CD20. CD79a expression detected in 23.8%(5/21) of the cases. Expression of CD56, CD27 and CD44v6 were 57.1% (12/21), 15.0% (3/20) and 23.8% (5/21), respectively. Follow-up data were available in 12 of the 21 patients (57.1%). Five patients were alive and 7 died. Three patients developed multiple myeloma (MM) and died of the tumor. SPB is a rare tumor with bone pain as the most common presenting symptom due to bone destruction. The diagnosis of EMP can only be established after exclusion of an extramedullay invasion by MM. Immunophenotype and IgH gene rearrangement analysis play important roles in the diagnosis of SPB.

Clonal loss of heterozygosity in microdissected Hodgkin and Reed-Sternberg cells.

The typical Hodgkin and ReedSternberg (HRS) cells are thought to represent the malignant cellular elements of Hodgkin’s disease (HD). The detection of immunoglobulin gene rearrangements in HRS cells indicates that they are clonally derived from B cells (1–8), but immunogenotyping, as such, does not provide any information on specific gene alterations possibly involved in the molecular pathology of HD. In many tumors, highly informative polymorphic DNA markers may identify loci harboring clonal loss of heterozygosity (LOH) and thus help to trace tumor suppressor genes (9,10). Although in HD, cytogenetic data suggest that nonrandom chromosomal deletions may occur at several loci, including 1q42, 4q26, and others, very little (if anything) is known about clonal LOH in HRS cells at the molecular level (11– 15). We, therefore, set out to study microdissected HRS cells from classical types of HD at candidate loci for LOH with a highly sensitive microsatellite polymerase chain reaction (PCR) assay. In seven patients with classical HD, HRS cells and surrounding cells including bystander lymphocytes were laser microdissected from formalin-fixed, paraffin-embedded tissue sections (Table 1). From a patient with nodularsclerosing HD (patient 6), samples were taken at presentation (6a) and at relapse (6b). In two patients (patient 6 and patient 7, a patient with mixed cellularitytype HD), frozen sections of lymphoma tissue yielded high-molecular-weight control DNA from microdissected cell populations. Buccal smears provided constitutional high-molecular-weight DNA in patients 5 and 6 (16,17). Patients gave their informed consent to include their material in this study. For molecular analyses, tetranucleotide repeat microsatellites were selected through the Genome Data Base at loci with a high frequency of chromosomal deletions in HD, which were predicted by the cytogenetic literature (11–15), and located at 1q42 (D1S517), 4q26 (D4S2301), 9p23 (D9S254), and 11q23 (D11S1294). Their PCR primer sequences are available in the Genome Data Base (http://www.gdb.org). A highly sensitive seminested PCR assay included a first round that used the primers indicated above. In a second PCR, one of the primers in each pair was replaced by an internal forward primer (Fint) or an internal reverse primer (Rin t ) : 1q42 (D1S517Rin t ) 5 8 CATGTGTCCATCAATGGTAG-38; 4q26 (D4S2301Fint) 58-GATGAGTGCTTAGACCATAGTA-38 ; 9p23 (D9S254Rint) 58-GTCTCCAATGCATGANCTT-38; and 11q23 (D11S1294Rint) 58-CTGGTTTGCTTTCCCTTTCTT-38 (software 4 “Primers! For the WWW”; http://www.williamstone.com) (Fig. 1, A). Allelic dropout during amplification of polymorphic microsatellite fragments may randomly affect either allele mimicking LOH. To exclude this pitfall, pooled samples of 10 purified HRS cells from a given patient were amplified (18,19). Ten picograms of DNA (corresponding to the DNA content of a single diploid cell) was consistently detected by our PCR assay. We also used a seminested PCR (1,3,20–22) to examine HRS cells from patients 3, 4, 6, and 7 for clonally rearranged immunoglobulin heavy-chain genes created through joining of variable, diversity, and joining immunoglobulin gene regions (V-D-J joining). The primers were a framework III immunoglobulin gene VH primer 58ACACGGC(C/T)(G/C)T(G/A)TATTACTGT-38 and a consensus JH primer (21) 58-ACCTGAGGAGACGGTGACC-38. The seminested primer was from VLJH sequences (58-GTGACCAGGT(N)CCTTGGCCCCA-38) (22) (Fig. 1, B). DNA extracted from buccal smears and from bystander lymphocytes showed constitutional individualspecific microsatellite patterns in all patients (Fig. 1, A). Of 259 samples of HRS cells microdissected from formalin-fixed tissue, 59 (23%) yielded a detectable microsatellite PCR product. Mock picks from tissue sections cleared of cellular material were consistently negative by PCR analysis. All patients except patient 5 reproducibly showed clonal LOH in HRS cells at one to three different microsatellite loci (Table 1). LOH was seen at all loci, but the locus most frequently altered was D4S2301 (4q26; four of five informative patients). High-molecular-weight DNA from fresh HRS cells and corresponding degraded DNA from formalin-fixed tissue yielded identical individual-specific microsatellite band patterns (Fig. 1, A). None of the HRS cell samples showed extra microsatellite bands (23). Patients 3, 4, 6, and 7 showed clonal immunoglobulin heavy-chain gene configurations in HRS cells and polyclonal patterns in bystander lymphoid cells (Fig. 1, B). In patient 6, a common rearranged immunoglobulin heavy-chain gene band in HRS cells, observed at presentation (6a) and at relapse (6b), indicated that the initial monoclonal population of HRS cells had relapsed. Our immunogenotype data from formalin-fixed HRS cells are in keeping with published data that HRS cells are mostly monoclonal B-cell populations (1–8,20,24). The detection of LOH has several implications in HD. The patients’ buccal mucosa and bystander lymphocytes showed constitutional microsatellite patterns indicating that LOH in HRS cells was an acquired specific genetic feature of HD. To the best of our knowledge, this is the first report on microsatellite PCR detection of clonal LOH in microdissected HRS cells. In contrast to our approach, classical cytogenetics permit the study of mitotic cells only and usually do not permit the identifi-

Utility of microdissection and polymerase chain reaction for the detection of immunoglobulin gene rearrangement and translocation in primary intraocular lymphoma

Objective Primary intraocular lymphoma, a non-Hodgkin’s lymphoma, is a primary central nervous system lymphoma (PCNSL). Diagnosis is usually made by identifying malignant, large B lymphocytes in the vitreous, eye, brain, and cerebral spinal fluid; however, these cells are few, friable, and difficult to recognize. Recently, clonal heavy chain immunoglobulin (IgH) gene rearrangement and bcl-2 gene translocation have been reported in systemic B-cell lymphoma and are used for the detection of malignant cells and in making a diagnosis. The authors investigated the molecular changes in three eyes and a chorioretinal biopsy specimen of four patients with PCNSL. Design Human tissue study. Materials Five ocular specimens of PCNSL were collected. Intervention The first patient had a diagnostic enucleation of the left eye. The second patient underwent diagnostic chorioretinal biopsy. In the third case, a pair of autopsied eyes with reactive lymphoplasmacytic infiltrates of a patient with acquired immune deficiency syndrome (AIDS) were studied. In the fourth case, an enucleated eye of a patient with AIDS-associated lymphoma was sampled. Main outcome measures The bcl-2 and IgH genes of the lymphoma cells from routine, paraffin-embedded, formaldehyde-fixed, or frozen histologic tissue sections were analyzed using microdissection and polymerase chain reaction (PCR) technique. Results Lymphoma cells obtained from the above four cases showed IgH rearrangement gene in the third framework of the V H region. Bcl-2-associated translocation also was detected in three cases (cases 1, 2, and 4). Conclusion Rearrangement of the IgH gene can serve as a molecular marker for PCNSL. Microdissection allows for procurement and analysis of specific, selected, minute cell populations that are obtained from histologic sections of the complex, heterogeneous tissue. Translocation of IgH and bcl-2, the apoptotic ”survival” signal and proto-oncogene, could contribute to the pathogenesis of PCNSL. The combination of microdissection and PCR is a powerful tool for studies of small lesions and cell populations and for understanding disease mechanisms.

Hairy cell leukemia: Treatment results and association with secondary malignancy

American Journal of HematologyVolume 48, Issue 4 p. 291-291 Letters and CorrespondenceFree Access Hairy cell leukemia: Treatment results and association with secondary malignancy Y. L. Kwong, Y. L. Kwong University Department of Medicine, Queen Mary Hospital, Hong KongSearch for more papers by this authorR. Liang, R. Liang University Department of Medicine, Queen Mary Hospital, Hong KongSearch for more papers by this authorE. Chiu, E. Chiu University Department of Medicine, Queen Mary Hospital, Hong KongSearch for more papers by this authorD. W. Chan, D. W. Chan University Department of Medicine, Queen Mary Hospital, Hong KongSearch for more papers by this authorT. K. Chan, T. K. Chan University Department of Medicine, Queen Mary Hospital, Hong KongSearch for more papers by this author Y. L. Kwong, Y. L. Kwong University Department of Medicine, Queen Mary Hospital, Hong KongSearch for more papers by this authorR. Liang, R. Liang University Department of Medicine, Queen Mary Hospital, Hong KongSearch for more papers by this authorE. Chiu, E. Chiu University Department of Medicine, Queen Mary Hospital, Hong KongSearch for more papers by this authorD. W. Chan, D. W. Chan University Department of Medicine, Queen Mary Hospital, Hong KongSearch for more papers by this authorT. K. Chan, T. K. Chan University Department of Medicine, Queen Mary Hospital, Hong KongSearch for more papers by this author First published: April 1995 https://doi.org/10.1002/ajh.2830480421Citations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 Kwong YL, Wong KF, Chan LC, Liang RHS, Chan JKC, Wei D, Chiu EKW, Chu YC, Todd D, Chan TK: The spectrum of chronic lymphoproliferative disorders in Chinese people-an analysis of 64 cases. Cancer 74: 174– 181, 1994. 2 Liang R, Chan V, Chan TK, Wong T, Chiu E, Lie A, Todd D: Detection of immunoglobulin gene rearrangement in lymphoid malignancies of B-cell lineage by seminested polymerase chain reaction gene amplification. Am J Hematol 43: 24– 28, 1993. 3 Machii T, Tokumine Y, Inoue R, Kitani T: Predominance of a distinct subtype of hairy cell leukemia in Japan. Leukemia 7: 181– 186, 1993. 4 Filleul B, Delannoy A, Ferrant A, Zeenebergh A, Bosly A, Doven C: A single course of 2-chlorodeoxyadenosine (2-CdA) does not eradicate leukemic cells in hairy cell leukemia (HCL) Patients achieving complete remission. Blood 82(suppl): 142a, 1993. 5 Kampmeier P, Spielberger R, Dickstein J, Mick R, Golomb H, Vardiman JW: Increased incidence of secondary neoplasms in patients treated with interferon α 2b for hairy cell leukemia: A clinicopathologic assessment. Blood 83: 2931– 6, 1994. Citing Literature Volume48, Issue4April 1995Pages 291-291 ReferencesRelatedInformation

Detection of immunoglobulin gene rearrangement in lymphoid malignancies of B‐cell lineage by seminested polymerase chain reaction gene amplification

Seminested polymerase chain reaction (PCR) was used to amplify the DNA fragments of the complementarity-determining region 3 of the immunoglobulin (Ig) gene heavy chain from the malignant cell specimens of patients with leukemias and lymphomas of B-cell lineage. Two different pairs of primers were used sequentially. Twenty of the 27 (74%) acute lymphoblastic leukemia (ALL) patients, 14 of 19 (74%) chronic lymphocytic leukemia (CLL) patients and eight of 20 (40%) non-Hodgkin's lymphoma (NHL) patients, who had rearrangement of the Ig gene heavy chain by Southern analysis, were positive by the seminested PCR. False-negative results appeared to occur more commonly in cases of lymphoma. The PCR analysis was also less likely to be positive if one-stage PCR studies with either pair of primers were both negative. The seminested PCR technique was found to have a high sensitivity of detecting malignant cells at the level of 0.02%. The clinical application of this assay needs to be investigated further.

Detection of immunoglobulin gene rearrangement in B-cell lymphomas by polymerase chain reaction gene amplification.

This is a report on our attempt to use polymerase chain reaction (PCR) to detect rearrangement of the immunoglobulin gene in the tissue specimens obtained from 30 patients with non-Hodgkin's lymphomas. There were 20 B-cell lymphomas and 10 T-cell. All 20 B-cell lymphomas but none of the 10 T-cell lymphomas had JH rearrangement by Southern analysis. Two pairs of primers (V670/OL-4 and VH26/OL-4) were designed to amplify the CDR3 region of the immunoglobulin gene heavy chain. The PCR analysis was positive using either one or both pairs of primers in 11 of the the 20 cases (55 per cent) of B-cell lymphomas which all had positive rearrangement by Southern analysis. The two pairs of primers seemed to produce complementary results as the specimens may be positive to one pair but negative to the other. The false negative rate of 45 per cent is however much higher than the respective figures of 18 per cent and 0 per cent observed in our patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia in a previous study. Peripheral blood and bone marrow biopsy specimens obtained at the time of initial diagnosis were available from 10 patients with B-cell lymphomas whose lymph node biopsy specimens at the time of diagnosis were positive by both Southern analysis and PCR. All these peripheral blood and marrow specimens had no microscopic evidence of involvement by lymphoma cells and JH rearrangement was not detected by Southern analysis. However, rearranged bands identical to that of the lymph node biopsy specimen were detected by PCR in the peripheral and marrow blood of one of them. This PCR technique has been shown to have a sensitivity of 0.1 per cent in our previous report and may be more useful than morphology alone or Southern analysis in detecting minimal lymphomatous involvement in the peripheral blood and bone marrow at the time of initial diagnosis. Further clinical correlation is required to confirm the finding.

Detection of immunoglobulin gene rearrangement in acute and chronic lymphoid leukemias of B-cell lineage by polymerase chain reaction gene amplification

Polymerase chain reaction (PCR) was used to amplify the DNA fragments of the complementarity determining region 3 of the immunoglobulin (Ig) gene heavy chain from the leukemic cell specimens of patients with acute and chronic lymphoid leukemias of B-cell lineage. Two different pairs of primers were tested. Fourteen of the 17 (82%) cases of acute lymphoblastic leukemia (ALL), and all 15 cases (100%) of B-cell chronic lymphocytic leukemia, who had rearrangement of the Ig gene heavy chain by Southern analysis, were positive by PCR with either one or both pairs of primers. This technique was able to detect leukemic cells at the level of 0.1%. Applying it to study the remission marrow specimens following induction chemotherapy was more useful than morphology alone in predicting early relapse of the leukemia.

Immunoglobulin gene rearrangement in multiple myeloma: Limitations of southern blot analysis

Thirty-six patients with early and advanced multiple myeloma were investigated with Southern blot analysis to determine the presence of immunoglobulin gene rearrangement as evidence of clonality. Rearrangements were not uniformly found, being detected in only 14 of 19 patients with newly diagnosed myeloma and in 15 of 17 cases of clinically advanced myeloma. A correlation between percentage of bone marrow plasma cells and detection of immunoglobulin gene rearrangement was noted; however, in four cases of early myeloma with > 10% marrow plasma cells, no rearrangement was found. These results suggest that Southern blot analysis may not be an optimal method for the determination of clonality in plasma cell dyscrasias or, alternatively, that a proportion of the plasma cells found on bone marrow examination in some patients with early myeloma may not be monoclonal.

Detection of Immunoglobulin Gene Rearrangement in B Cell Neoplasias by Polymerase Chain Reaction Gene Amplification.

(1991). Detection of Immunoglobulin Gene Rearrangement in B Cell Neoplasias by Polymerase Chain Reaction Gene Amplification. Leukemia & Lymphoma: Vol. 5, No. 1, pp. 9-22.

Detection of immunoglobulin gene rearrangement in B lymphoid malignancies by polymerase chain reaction gene amplification

Immunoglobulin heavy chain gene rearrangement serves as a marker of cell lineage and clonality in B lymphoproliferative disorders. We have used polymerase chain reaction (PCR) gene amplification to detect immunoglobulin gene rearrangements involving VH251, a heavy chain variable region preferentially utilized in B lymphoproliferative disorders. Using synthetic amplimers derived from VH251 and the heavy chain joining region, under conditions of high stringency, a homogeneous VH251-specific fragment of approximately 350 bp could be amplified from leukaemic DNA. Of 53 cases of B lineage acute lymphoblastic leukaemia screened for VH251 rearrangement by PCR, 10 were positive. A background level of VH251 rearrangement could also be amplified from normal peripheral blood and bone marrow DNA, but a VH251 rearranged leukaemic clone representing 0.01% of bone marrow mononuclear cells could be readily detected. The application of PCR to detect immunoglobulin gene rearrangement involving VH251 and potentially other preferentially utilized V regions provides a sensitive method both for tracking malignant B cells and for the study of normal B cell developmental pathways through which B lineage malignancies arise.

Immunoglobulin gene rearrangement as a diagnostic criterion of B-cell lymphoma.

We describe the use of the Southern blot hybridization technique to diagnose B-cell lymphoma by detecting clonal immunoglobulin gene rearrangements in lymph node and other biopsy tissues. DNA was isolated from a wide variety of neoplastic and non-neoplastic specimens and analyzed for the presence of rearranged immunoglobulin genes using radiolabeled DNA probes specific for the heavy- and light-chain immunoglobulin constant region genes. Among the specimens examined, clonal immunoglobulin gene rearrangements were found only in biopsy samples of B-cell lymphoma and not in samples containing reactive lymphoid processes or non-B-cell cancers. In lymphomas, the presence of rearrangements for either the kappa or lambda light-chain gene correlated with expression of one or the other of these chains when cellular immunoglobulins could be detected by frozen-section immunophenotyping techniques. The analysis of immunoglobulin gene rearrangements offers several advantages over conventional diagnostic methods for lymphomas, including improved sensitivity in detecting minor populations of neoplastic lymphocytes composing as little as 1% of the total cell population. In addition, clonal immunoglobulin gene rearrangements are demonstrable in a subset of lymphomas that lack detectable surface or cytoplasmic immunoglobulin, thus offering positive evidence for both malignancy and the B-cell origin of these tumors. Our studies indicate that detection of immunoglobulin gene rearrangements is a valuable method for diagnosis and classification of various lymphoproliferative disorders that are difficult to evaluate histologically or that lack distinctive antigenic markers.