Clonal Immunoglobulin Heavy Chain Rearrangements Research Articles

Evaluation of next-generation sequencing versus next-generation flow cytometry for minimal-residual-disease detection in Chinese patients with multiple myeloma

PurposeTo evaluate the efficacy of next-generation sequencing (NGS) in minimal-residual-disease (MRD) monitoring in Chinese patients with multiple myeloma (MM).MethodsThis study analyzed 60 Chinese MM patients. During MRD monitoring in these patients’ post-therapy, clonal immunoglobulin heavy chain (IGH) rearrangements were detected via NGS using LymphoTrack assays. MRD monitoring was performed using NGS or next-generation flow cytometry (NGF), and the results were compared. Additionally, the sensitivity and reproducibility of the NGS method were assessed.ResultsThe MRD detection range of the NGS method was 10–6–10–1, which suggested good linearity, with a Pearson correlation coefficient of 0.985 and a limit of detection of 10–6. Intra- and inter-assay reproducibility analyses showed that NGS exhibited 100% reproducibility with low variability in clonal cells. At diagnosis, unique clones were found in 42 patients (70.0%) with clonal IGH rearrangements, which were used as clonality markers for MRD monitoring post-therapy. Comparison of NGS and NGF for MRD monitoring showed 79.1% concordance. No samples that tested MRD-positive via NGF were found negative via NGS, indicating the higher sensitivity of NGS. MRD could be detected using NGS in 6 of 7 samples before autologous hematopoietic stem-cell transplantation, and 5 of them tested negative post-transplantation. In contrast, the NGF method could detect MRD in only 1 sample pre-transplantation.ConclusionCompared with NGF, NGS exhibits higher sensitivity and reproducibility in MRD detection and can be an effective strategy for MRD monitoring in Chinese MM patients.

The Effect of Preemptive Rituximab Treatment in Patients with EBV Viremia after Solid Organ Transplantation on the Development of Post-Transplant Lymphoproliferative Disorder

The association between Epstein-Barr Virus (EBV) reactivation after solid organ transplantation (SOT) and Post-Transplant Lymphoproliferative Disorder (PTLD) is well described. There are currently no guidelines regarding the use of rituximab preemptively in patients with EBV viremia after SOT to reduce the risk of PTLD. We sought to determine whether rituximab in SOT patients with EBV viremia has benefit in preventing PTLD. This retrospective analysis of electronic medical record data identified SOT recipients who experienced EBV viremia post-transplant between January 2010 and February 2023 at Columbia University Irving Medical Center (CUIMC). Data queries were clinically reviewed. Logistic regression analysis was performed to investigate the relationship between predictor variables including rituximab administration, log-transformed EBV peak titer, duration of EBV viremia, and time from SOT to EBV reactivation, with the binary primary outcome of PTLD diagnosis status. The analysis encompassed both univariate and multivariate logistic regression. There were 2,168 patients who met the inclusion criteria of having EBV reactivation after SOT, out of 6,395 total SOT performed at CUIMC during this timeframe. Patient characteristics are described in table 1. The median peak EBV titer was 154 (range: not quantifiable - 5,335,816), the median duration of EBV viremia was 78 days (range: 1 - 6,980), and the median time from SOT to EBV reactivation was 688 days (range: 0-11103). Of the 2,168 patients, 185/2168 (8.5%) received rituximab for indications other than PTLD including EBV viremia, treatment of rejection, antibody desensitization therapy/immunosuppression peri-transplant, and other non-transplant related indications (e.g. lupus nephritis). Of the patients who received rituximab, 182/185 (98.4%) did not develop PTLD. The 3/185 (1.6%) patients who did develop PTLD were all ≤ 20 years-old at time of diagnosis, and had EBER(+), monomorphic PTLD, diffuse large B-cell lymphoma type, with a non-germinal center phenotype, involving the gastrointestinal tract. Two were CD20 positive, all 3 had partial weak CD30 positivity, and all 3 had clonal immunoglobulin heavy chain rearrangements. Among the patients who did not receive preemptive rituximab post-SOT, 195/1983 (9.8%) developed PTLD, whereas 1788/1983 (90.2%) did not. Prior rituximab therapy for any indication in patients with EBV viremia post-SOT was associated with lower risk of subsequent PTLD by univariate analysis (0.14% vs 8.39%, OR 0.15, p = 0.0013, 95% CI 0.048-0.48). Receipt of rituximab (OR 0.12, p=0.0002), log-transformed peak EBV titer (OR 1.10, p<0.0001), and duration of EBV viremia in months (OR 1.01, p<0.0001) were all independently associated with risk for PTLD by multivariate analysis (figure 1). The relationship between time from SOT to EBV viremia was not significantly associated with development of PTLD. This retrospective analysis suggests that administering preemptive rituximab in SOT patients with EBV viremia may lower the risk of developing PTLD. The fact that peak EBV titer and duration of EBV viremia remain associated with PTLD in multivariate analyses suggests that the benefit of rituximab may be independent of the degree or duration of EBV viremia. A prospective analysis of factors such as area under the curve of EBV exposure, HLA status, T cell clonotype diversity, organ type, and immunosuppression type, is needed to better understand individual risks for improved selection of those who would benefit from preemptive rituximab.

Combining MYD88 L265P mutation detection and clonality determination on CSF cellular and cell-free DNA improves diagnosis of primary CNS lymphoma.

Diagnosis of primary central nervous system lymphoma (PCNSL) is challenging, and although brain biopsy remains the gold standard, cerebrospinal fluid (CSF) constitutes a less invasive source of lymphomatous biomarkers. In a retrospective cohort of 54 PCNSL cases tested at diagnosis or relapse, we evaluated the contribution of immunoglobulin heavy chain (IGH) gene clonality and MYD88 L265P detection on both CSF cell pellets and supernatants, in comparison with cytology, flow cytometry, interleukin (IL)-10 and IL-6 quantification. Clonality assessment included a new assay to detect partial IGH-DJ rearrangements. Clonal IGH rearrangements and/or MYD88 L265P mutation were detected in 27 (50%) cell pellets and 24 (44%) supernatant cell-free (cf) DNA. Combining analyses on both compartments, 36 (66%) cases had at least one detectable molecular marker, present only in cfDNA for 9 (16%) of them. While cytology and flow cytometry were positive in only 7 (13.0%) and 9 (17.3%) cases respectively, high IL-10 levels were observed in 36 (66.7%) cases. Overall, taking into account molecular and cytokine results, 46/54 (85%) cases had at least one lymphomatous biomarker detectable in the CSF. These results show that this combination of biomarkers evaluated on both cell pellet and supernatant CSF fractions improves significantly the biological diagnosis of PCNSL.

Targeted Locus Amplification (TLA): A Novel Next Generation Sequencing (NGS) Technology to Detect New Molecular Markers and Monitoring Minimal Residual Disease (MRD) in Mantle Cell and Follicular Lymphoma

Background: Minimal residual disease (MRD) monitoring by PCR methods is a strong and standardized predictor of clinical outcome in mantle cell (MCL) and follicular (FL) lymphoma [Pott C., Blood 2010; Ladetto M, Blood 2013]. However, some technical limitations hamper its feasibility in daily clinical routine. First of all, current techniques allow the identification of a molecular marker only in 75-80% of MCL and 55-60% of FL patients. All the other “no marker” cases cannot be studied for MRD purposes. Next generation sequencing (NGS) tools might overcome these limitations. Among them, the recently developed targeted locus amplification (TLA) technology [de Vree P.J., Nat Biotechnol 2014] that selectively amplifies and sequences entire genes on the basis of the crosslinking of physically proximal DNA loci, was able to identify novel candidate oncogenes and gene fusions in acute lymphoblastic leukemia. [Kuiper R.P.; Vroegindeweij EM, 57th ASH Communication, Blood 2015].The aim of this study was to test the TLA approach in MCL and FL diagnostic samples, with at least one translocation confirmed by FISH and a failure in molecular marker identification, with the aim to increase the rate of success in marker screening for MRD purposes. Moreover, the performances of the newly TLA molecular markers were compared to the standardized quantitative PCR (qPCR) approach for MRD monitoring.Methods: Genomic DNA was extracted from bone marrow (BM) and peripheral blood (PB) samples of MCL and FL patients, enrolled in prospective clinical trials of the Fondazione Italiana Linfomi (FIL). All patients provided written informed consent for the research use of the biological samples. Libraries for TLA (Cergentis, Utrecht) were prepared using only one couple of antisense primers targeting the immunoglobulin heavy chain (IGH) enhancer locus, as previously described [Hottentot QP, Methods Mol Biol. 2017], and sequenced on MiSeq platform (Illumina, San Diego). MRD monitoring was carried out by qPCR designing allele specific oligonucleotides (ASO) and consensus probes based on the new breakpoint sequences obtained from TLA NGS. Finally, the efficiency of TLA novel markers to monitor MRD was compared to MRD data obtained from clonal IGH rearrangements detected and tracked by standardized ASO qPCR. [Van der Velden VHJ, Leukemia 2007].Results: TLA was firstly tested on 21 t(11;14)-positive, BCL-1/IGH major translocation cluster (MTC) negative MCL baseline samples (8 BM and 13 PB): in 86% of cases (18/21) a novel BCL-1/IGH breakpoint was identified by NGS. In these cases, TLA described new rearrangements between “no-MTC” BCL1 loci and JH1, JH4, JH5 or JH6 regions on chromosome 14. Therefore, additional 8 FL baseline BM samples, resulting t(14;18) FISH positive and BCL-2/IGH MBR/mcr/“minor rearrangements”-negative by PCR, were tested obtaining a 62% (5 out of 8 samples) success rate. Also in these cases, new and not yet described BCL2/IGH rearrangements were found. Additionally, to assess the specificity of the technology, the MBR-positive control sample (DOHH2 cell line) was tested and correctly sequenced, as well. Interestingly, among the new TLA positive FL cases, in one sample, the NGS technology highlighted the breakpoint on the DH region of chromosome 14, describing an unusual t(14;18) translocation. 5/29 cases (17%) in which TLA was not able to identify a new marker were characterized by a low tumor infiltration, mainly <5% by flow cytometry (median 4.4%, range 0.03-8%). Secondly, a validation of the NGS sequences was performed by ASO primers designed on the new TLA breakpoints and MRD was monitored by ASO qPCR. Overall, in 14 MCL cases where also a classic IGH-based marker was available, the MRD results were superimposable between the two markers (Fig1A), with good and overlapping performances in terms of overall correlation (r2=0,86; Fig 1B). In FL samples, ASO qPCR confirmed the high tumor burden of the analyzed tissues (Fig 1C). Finally, an extension of the analyzed samples series and MRD validation is currently ongoing among additional FL patients.Conclusion: The TLA NGS technology allowed to identify a translocation-based novel molecular marker in FISH positive t(11;14) and t(14;18) MCL and FL baseline samples, where the classical Sanger sequencing failed a marker identification. These new breakpoints can reliably be used for the design of ASO qPCR primers for MRD detection in previously not traceable patients [Display omitted] DisclosuresKlous:Cergentis BV: Employment. Yilmaz:Cergentis BV: Employment. Vitolo:Gilead: Honoraria; Mundipharma: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria. Luminari:PFIZER: Speakers Bureau; GILEAD: Speakers Bureau; TEVA: Membership on an entity's Board of Directors or advisory committees; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees; ROCHE: Membership on an entity's Board of Directors or advisory committees; TAKEDA: Membership on an entity's Board of Directors or advisory committees. Federico:takeda: Honoraria, Research Funding. Splinter:Cergentis BV: Employment.

Genomics of Limited-Stage Diffuse Large B-Cell Lymphoma Developing Late Relapse: Analysis of Gene Alterations in Paired Primary and Late Relapsed Tumors By Target Sequencing

Genomics of Limited-Stage Diffuse Large B-Cell Lymphoma Developing Late Relapse: Analysis of Gene Alterations in Paired Primary and Late Relapsed Tumors By Target Sequencing

Next generation sequencing of the clonal IGH rearrangement detects ongoing mutations and interfollicular trafficking in in situ follicular neoplasia.

Follicular lymphoma (FL) is characterized genetically by a significant intraclonal diversity of rearranged immunoglobulin heavy chain (IGH) genes and a substantial cell migration activity (follicular trafficking). Recently, in situ follicular neoplasia (ISFN), characterized by accumulations of immunohistochemically strongly BCL2-positive, t(14;18)+ clonal B cells confined to germinal centers in reactive lymph nodes, has been identified as a precursor lesion of FL with low risk of progression to manifest FL. The extent of ongoing somatic hypermutation of rearranged IGH genes and interfollicular trafficking in ISFN is not known. In this study we performed an in depth analysis of clonal evolution and cell migration patterns in a case of pure ISFN involving multiple lymph nodes. Using laser microdissection and next generation sequencing (NGS) we documented significant intraclonal diversity of the rearranged IGH gene and extensive interfollicular migration between germinal centers of the same lymph node as well as between different lymph nodes. Furthermore, we identified N-glycosylation motifs characteristic for FL in the CDR3 region.

Detection of Immunoglobulin Heavy Chain Gene Clonality by Next-Generation Sequencing for Minimal Residual Disease Monitoring in B-Lymphoblastic Leukemia.

Minimal residual disease (MRD) following B-lymphoblastic leukemia (B-ALL) treatment has gained prognostic importance. Clonal immunoglobulin heavy chain (IGH) gene rearrangement is a useful follow-up marker in B-ALL owing to its high positivity rate. We evaluated the performance and clinical applicability of a next-generation sequencing (NGS) assay for IGH rearrangement in B-ALL MRD monitoring. IGH rearrangement was tested by using fluorescence PCR-fragment analysis and the NGS assay in eight B-ALL patients. The NGS assay was run on two platforms: the Ion Torrent PGM (Thermo Fisher Scientific, USA) (18 samples from 1st to 7th patients) and the MiSeq system (Illumina, USA) (four samples from 8th patient). All initial diagnostic samples and four follow-up samples were positive for clonal IGH rearrangement with fluorescence PCR-fragment analysis and the NGS assay, and six follow-up samples were positive only with NGS. In one case with BCR-ABL1 translocation, BCR-ABL1 quantitative PCR was negative but the NGS IGH assay was positive just prior to full-blown relapse, suggesting the high sensitivity and clinical utility of the NGS assay. The NGS assay is proposed for MRD monitoring in B-ALL Additional studies are needed to confirm the clinical implications of cases showing positive results only in NGS.

PCR Analysis of IgH and TCR-γ Gene Rearrangements as a Confirmatory Diagnostic Tool for Lymphoproliferative Disorders.

This study investigates PCR analysis of immunoglobulin heavy chain (IgH) and T cell receptor (TCR) gene rearrangements on paraffin-embedded tissue sections and bone marrow aspirates of patients suspected to have lymphoproliferative disorders but with inconclusive diagnosis in histopathological examination. 130 samples of patients with inconclusive immunohistochemistry results were evaluated for clonal rearrangement of IgH and TCR genes. Based on histopathology examination, the patients were divided into three groups: the first group without any definite diagnosis of lymphoproliferative disorders (60 cases, 46.2%), the second group suspected to have a lymphoproliferative disorder but in favor of benign disorders (19 cases, 14.6%) and the third group suspect to lymphoproliferative disorders but relatively in favor of malignant disorders (51 cases, 39.2%). After DNA extraction and quality control, semi-nested PCR was performed using consensus primers for amplification of TCR-γ and CDR-3 regions of IgH genes. PCR products were analyzed after heteroduplex analysis using polyacrylamide gel electrophoresis, and were subject to silver staining. Totally, in over half of the cases (55.4%), a monoclonal pattern was found in IgH or TCR-γ genes rearrangements. Monoclonal IgH gene rearrangement was detected in 48.1% of patients, whereas monoclonal TCR-γ gene rearrangement was found in 33.6% of them, which was not statistically significant (P=0.008). Only in 32 patients (24.6%) were the results of TCR-γ and IgH gene rearrangements consistent with respect to the presence (2.3%) or absence (22.3%) of monoclonality. Finally, PCR analysis of TCR-γ and IgH gene rearrangements led to definite diagnosis in 105 patients (80.8%), and only 25 cases (19.2%) remained inconclusive. Our results emphasize the usefulness of gene rearrangement study in cases without a definite diagnosis in immunohistochemistry studies. Multiple PCR analysis results when combined with patient's clinical course and immunohistochemistry can lead to early diagnosis and subsequent therapy.

Kaposi sarcoma herpesvirus/human herpesvirus‐8–negative effusion‐based lymphoma: Report of 3 cases and review of the literature

Primary effusion lymphoma (PEL) is a rare subtype of large B-cell lymphoma that arises in body cavities without detectable tumor masses. PEL is universally associated with Kaposi sarcoma herpesvirus (KSHV)/human herpesvirus-8 (HHV8). Despite overlapping features, KSHV/HHV8-negative effusion-based lymphoma is a distinct entity from PEL. To date, 52 cases have been reported. The authors report 3 additional cases received in their laboratory from 2007 to 2012. Clinical data, cytomorphologic features, and immunophenotypic features of the 3 cases were described and compared with those reported in the literature. The cells in HHV8-negative effusion lymphoma commonly revealed large cell, immunoblastic morphology and B-cell immunophenotype. The 3 cases demonstrated cytomorphologic and immunophenotypic variability. Cytomorphologically, 1 case contained large, highly atypical cells with a moderate amount of cytoplasm, round nucleus, coarsely granular chromatin, and a single macronucleolus. The other 2 cases had medium to large atypical cells with high nuclear-to-cytoplasmic ratios, slightly irregular to cleaved nuclei, and multiple conspicuous nucleoli. One case had a null phenotype with aberrant cytokeratin expression. B-cell phenotype was established by clonal immunoglobulin heavy-chain rearrangement using polymerase chain reaction, whereas the other 2 cases demonstrated a B-cell phenotype by flow cytometry and immunohistochemical staining. All 3 cases were negative for both HHV8 and Epstein-Barr virus. HHV8-negative effusion lymphoma exhibits clinical, cytomorphologic, and immunophenotypic variability. Cases with a null-phenotype can be particularly challenging. When effusion lymphoma is suspected, ancillary tests are helpful. Moreover, HHV8 detection is critical in differentiating PEL and HHV8-negative effusion lymphoma, because they have overlapping features yet different prognoses.

Massive evolution of the immunoglobulin heavy chain locus in children with B precursor acute lymphoblastic leukemia

AbstractThe ability to distinguish clonal B-cell populations based on the sequence of their rearranged immunoglobulin heavy chain (IgH) locus is an important tool for diagnosing B-cell neoplasms and monitoring treatment response. Leukemic precursor B cells may continue to undergo recombination of the IgH gene after malignant transformation; however, the magnitude of evolution at the IgH locus is currently unknown. We used next-generation sequencing to characterize the repertoire of IgH sequences in diagnostic samples of 51 children with B precursor acute lymphoblastic leukemia (B-ALL). We identified clonal IgH rearrangements in 43 of 51 (84%) cases and found that the number of evolved IgH sequences per patient ranged dramatically from 0 to 4024. We demonstrate that the evolved IgH sequences are not the result of amplification artifacts and are unique to leukemic precursor B cells. In addition, the evolution often follows an allelic exclusion pattern, where only 1 of 2 rearranged IgH loci exhibit ongoing recombination. Thus, precursor B-cell leukemias maintain evolution at the IgH locus at levels that were previously underappreciated. This finding sheds light on the mechanisms associated with leukemic clonal evolution and may fundamentally change approaches for monitoring minimal residual disease burden.

Clinicopathological analysis of a composite lymphoma containing both T‐ and B‐cell lymphomas

Composite lymphomas (CLs) have been reported in 1-4.7% of all lymphomas, however, CLs containing both T- and B-cell lymphomas (CTBLs) are very rare. Here, we examined the clinical and pathological features of 29 CTBLs. These CTBLs included 21 patients with angioimmunoblastic T-cell lymphoma (AITL) and diffuse large B-cell lymphoma (DLBCL), two with adult T-cell leukemia/lymphoma and DLBCL, one with AITL and Follicular lymphoma, one with Lennert lymphoma and DLBCL, one with subcutaneous panniculitis-like T-cell lymphoma and DLBCL, one with peripheral T-cell lymphoma (PTCL) and DLBCL, one with cutaneous T-cell lymphoma and DLBCL, and one with PTCL and chronic lymphocytic leukemia. Eighteen of 27 patients (67%) were shown to be Epstein-Barr virus (EBV)-encoded RNA-positive in their B-cell lymphoma component. T-cell and B-cell clonality were confirmed by flow cytometry, Southern blot analysis, and/or polymerase chain reaction (PCR). Using Southern blot analysis, clonal immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) rearrangements were detected in 11 of 21 and 15 of 24 cases, respectively. Using PCR analysis, clonal IgH and TCR rearrangements were detected in 7 of 8 and 7 of 7 Southern blot-negative cases, respectively. Our results suggested that PCR analysis was useful in diagnosing CTBL.

Molecular Characterization of Immunoglobulin Gene Rearrangements in Diffuse Large B-Cell Lymphoma: Antigen-Driven Origin and IGHV4-34 as a Particular Subgroup of the Non-GCB Subtype

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34, IGHV3-23, and IGHV4-39, accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell-like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.

Florid Reactive Lymphoid Hyperplasia of the Lower Female Genital Tract (Lymphoma-like Lesion): A Benign Condition That Frequently Harbors Clonal Immunoglobulin Heavy Chain Gene Rearrangements

Lymphoma-like lesions (LLL) of the lower female genital tract are florid reactive inflammatory processes that mainly occur in women in their reproductive years. Histologically, they are characterized by a dense lymphoid infiltrate with admixed large cells that is often suspicious for lymphoma. In contrast to lymphoma, however, they are superficial lesions that typically show surface erosion and a mixed lymphoid infiltrate and do not have evidence of a mass, deep invasion, or prominent sclerosis. With the advent of widespread molecular genetic testing, it would seem that LLLs should be polyclonal helping make the correct diagnosis. However, we have found cases with morphologic and immunophenotypic features of LLLs and evidence of clonal rearrangement of the immunoglobulin heavy chain (IGH) gene, potentially leading to misdiagnosis. We examined the clinicopathologic features and outcome of 12 patients with LLL (9 in the cervix and 3 in the endometrium). The patients ranged in age from 18 to 54 (median 37) years and came to medical attention because of squamous dysplasia (8 patients), vaginal bleeding (3), or adnexal mass (1). One patient had an endocervical polyp, but otherwise none had a discrete mass. The specimens contained a dense, polymorphous inflammatory infiltrate, commonly associated with mucosal erosion. Immunohistochemical studies showed a mixture of B and T cells without immunoglobulin light chain restriction. Four cases (all cervical) had a clonal IGH gene rearrangement by PCR. There was no evidence of lymphoma on staging or on follow-up in any patient, including the 4 patients with clonal IGH rearrangement, after a mean follow-up of 3.5 years (range: 4 mo to 13 y). In summary, we describe 12 patients with LLL of the lower female genital tract. Four of the 9 cases (44%) analyzed by PCR had a clonal IGH gene rearrangement. The clinical and pathologic features of these cases suggest that a clonal IGH rearrangement in this setting does not warrant a diagnosis of lymphoma. Careful correlation of clinical, histologic, immunophenotypic, and genetic features is required to avoid misdiagnosis and inappropriate treatment. Routine microscopic findings and detailed clinical information remain paramount in establishing the correct diagnosis.

Monoclonal B cells detected in autologous PBSC grafts from patients with classical Hodgkin lymphoma: impact on relapse and survival following transplantation

Autologous peripheral blood stem cell transplantation (PBSCT) for Hodgkin lymphoma (HL) is curative for many patients with relapsed or refractory disease. Relapsing disease, however, remains a major problem. Neoplastic transformation of B-lymphocytes probably underlies the development of classical HL. Whether clonal B cells are critical for disease evolution and response to therapy in HL remains uncertain. We investigated the impact of clonal B cells detected in peripheral blood stem cell (PBSC) collections on the outcome of patients with HL undergoing transplant. Qualitative semi-nested PCR was carried out on genomic DNA from mononuclear cells from PBSCs to determine the presence of clonal immunoglobulin heavy chain (IgH) complementary-determining region 3 (CDR3) gene rearrangements. Clinical factors were assessed for their association with relapse, overall survival (OS) and progression-free survival (PFS). Among 39 patients undergoing PBSCT, 12 grafts (31%) were PCR positive for clonal IgH rearrangements. OS was better in the PCR-negative group (logrank test, P=0.041). The OS at 5 years was 81% in PCR-negative versus 39% in PCR-positive patients; hazard ratio was 3.23 (95% confidence interval: 0.98-10.63). There was a trend towards better PFS (logrank test, P=0.12), estimated as 71% at 5 years in PCR-negative versus 41% in PCR-positive patients. Clonal B-lymphocytes in PBSC collections of patients with HL identify patients at risk of poor outcome. Larger series are needed to confirm our observations. Insight regarding the role of monoclonal B cells may lead to improved therapies.

Clinical significance in detection of immunoglobulin heavy chain clonal rearrangement in bone marrow of patients with B cell lymphoma

To explore the feasibility of semi-nested PCR technique for detection of immunoglobulin heavy chain (IgH) clonal rearrangement in bone marrow of B-cell lymphoma patient and to further evaluate its clinicopathological value. Gene clonal rearrangement of IgH was detected by semi-nested PCR using primers of FR2 & FR3A in 105 bone marrow samples of patients with B-cell lymphoma. The PCR detection results were compared with the cytomorphology of bone marrow aspiration biopsy. The correlation between PCR detection results and clinicopathological factors were evaluated. Among 105 cases of B-cell lymphoma, bone marrow involvement was detected by PCR technique in 48 cases (45.7%), while only 22 cases (21.0%) were detected by bone marrow cytological analysis. There was a significant difference between two methods (P < 0.05), and the concordance rate was 71.4%. The incidence of bone marrow involvement at the time of initial diagnosis detected by PCR technique was 30.8% for diffuse large B cell lymphoma (DLBCL), 25.0% for follicular lymphoma (FL), and 100.0% for small lymphocytic lymphoma (SLL), respectively. Bone marrow involvement detected by PCR detection correlated with Ann Arbor stage. Rate of clonal IgH gene rearrangement by PCR in early B-cell lymphoma was lower than that in advanced stage B-cell lymphoma patients (P = 0.02). There was no statistically significant difference in efficacy between patients with positive and negative results detected by PCR (P > 0.05). But difference in complete response (CR) rate (23.3% and 46.3%) had significant difference (P = 0.019). Semi-nested PCR analysis may be an effective method for detection of abnormalities in bone marrow in patients with B-cell lymphoma and is superior to cytomorphology. The positive rate in patients with advanced Ann Arbor stage is higher than that in patients with early Ann Arbor stage, and patients with PCR negative result have more chances to achieved CR after treatment.

Detection of Clonal IGH Gene Rearrangements: Summary of Molecular Oncology Surveys of the College of American Pathologists

The diagnosis of B-cell lymphoid malignancy can frequently be substantiated by detecting clonal immunoglobulin heavy chain (IGH) gene rearrangements, which is typically done by polymerase chain reaction (PCR) amplification and/or Southern blot analysis. To characterize current laboratory practice for the assessment of IGH rearrangements and to identify opportunities for improvement. The data from the Molecular Oncology Proficiency Survey distributed to participating laboratories by the Molecular Pathology Committee of the College of American Pathologists from 1998 through 2003 were analyzed. Thirty-nine proficiency survey specimens (29 positive and 10 negative for clonal IGH rearrangements) were distributed. For Southern blot analysis, 944 results were reported, with a successful response rate of 95%. For PCR detection, 2349 results were reported, with a successful response rate of 72%. A higher rate of successful responses by PCR was achieved using framework 3 primers in combination with other frameworks (82%) compared with framework 3 primers only (76%) and when fresh/frozen (72%) compared with paraffin-embedded (65%) tissues were analyzed. The performance of the participating laboratories was very good, by both Southern blot and PCR analysis. As expected, Southern blot analysis consistently detects a higher proportion of IGH rearrangements than PCR analysis. Further improvement and standardization of the IGH PCR assay is important if it is to replace Southern blot analysis as the standard method. Participation in this survey is a valuable tool for assessing laboratory performance and it directs our attention to areas where we may improve laboratory practice.

Detection of rearranged immunoglobulin DNA in plasma

17518 Background: Tumor DNA is increasingly recognized as a marker in plasma or serum. B cells are characterized by immunoglobulin heavy chain rearrangements (IgH-R)and B cell malignancies by clonal IgH-R. We hypothesized that detection of clonal IgH-R DNA (a DNA spike) might reflect the presence of malignancy. Methods: We used a PCR and capillary electrophoresis. DNA from plasma of 6 healthy donors, 5 patients treated with rituximab, 5 persons with AIDS (PWA) but not lymphoma, 5 patients with AIDS-related lymphoma (ARL) and 5 patients with Hodgkin’s lymphoma (HL) was studied. In some cases matched peripheral blood mononuclear cells (PBMC) and followup specimens were available. Results: In plasma from healthy donors there was a normal (bell-shaped) distribution of IgH-R. In patients treated with rituximab (including a patient with an autoimmune disorder but no malignancy), IgH-R in plasma decreased by at least 100-fold. In PWA, there was a normal distribution of IgH-R. In pretreatment plasma from 2 of 5 patients with ARL, a DNA spike was detected superimposed on the background of IgH-R. The DNA spike was not detected in PBMC. In both ARL patients the spikes disappeared with therapy. However, in 1 it reappeared, and shortly thereafter the patient relapsed. Clinical relapse was associated with an increase in spike amplitude. The spike disappeared again with salvage chemotherapy. Identity of the pretreatment and relapse spikes was confirmed by sequencing. In 1 of 5 patients with HL a pretreatment DNA spike was detected in plasma. It decreased in serial specimens during the first month of combination chemotherapy. Conclusions: A normal distribution of rearranged Ig DNA is readily detected in plasma from healthy donors and patients. This normally distributed rearranged Ig DNA consistently disappears with rituximab therapy. A DNA spike can be detected in the plasma of some patients with B lineage lymphoma even when that spike is not evident in PBMC consistent with the possibility that this DNA is not derived from circulating tumor cells. The reappearance of a DNA spike may presage relapse. Further investigation will be required to determine the sensitivity and specificity of detection of such Ig DNA spikes and to confirm a tumor origin. No significant financial relationships to disclose.

Canine Indolent Nodular Lymphoma

Sixty-six cases of indolent canine lymphoid proliferation were reviewed. Age ranged from 1.5 to 16 years (median 9.0 years). Dogs of 26 breeds, plus 13 of mixed breeding or unknown lineage, were represented. B-Cell lymphomas (CD79a+) predominated. Marginal zone lymphoma (MZL), the largest group, involved lymph node (33 cases) and spleen (13 cases), with both tissues involved in five of these cases. Follicular lymphoma (FL) involved lymph nodes (five cases), and mantle cell lymphoma (MCL) occurred as solitary splenic masses (three cases). Nodal CD3+ T-zone lymphomas (TZL) (10 cases), were included since they resembled late-stage MZL at the architectural level. Two cases of marginal zone hyperplasia (MZH) were included to aid in differentiation of early MZL. Clonality status was determined in 54 cases by analysis of immunoglobulin heavy chain (IGH) and T-cell antigen receptor gamma (TCRG) gene rearrangement. Clonal rearrangement of IGH was detected in 28 of 35 MZL cases (80%), four of four FL cases (100%) and three of three MCL cases (100%). Concurrent cross lineage rearrangement of TCRG was detected in six MZL and two FL cases. Clonal rearrangement of TCRG was documented in five of eight TZL cases (63%). Limited survival data obtained for 18 dogs indicated that the B-cell lymphomas (MZL, MCL, and FL) and the T-cell lymphoma (TZL) were associated with indolent behavior and long survival. Although to the authors' knowledge, the true incidence of canine indolent lymphomas is unknown, the tumors are not rare and may have been underrecognized. Recognition of their architectural features, routine application of immunophenotyping, and molecular clonality assessment should alleviate this.

Analysis of clonal immunoglobulin heavy chain rearrangements in ocular lymphoma

The morphologic diagnosis of primary and metastatic intraocular lymphoma (IOL) was made difficult by the paucicellular specimens with fragile populations of lymphocytes retrieved through pars plana vitrectomy (PPV). The analysis of immunoglobulin heavy chain (IgH) gene rearrangements (AIGHR) was used as an adjunct to cytopathology and flow cytometry in systemic lymphoma. In IOL, the sensitivity and specificity of AIGHR are unknown. The authors reviewed the clinical records of patients who underwent PPV for suspicion of IOL at the Immunology and Uveitis Service of the Massachusetts Eye and Ear Infirmary (Boston, MA) between 2000 and 2002. AIGHR was performed as a routine diagnostic test on cell lysates isolated from < 0.5 mL of vitreous fluid. The authors used seminested polymerase chain reaction (PCR) with consensus primers for the VDJ-region of the IgH gene. PCR products were analyzed by polyacrylamide gel electrophoresis. Thirty patients (37 specimens) with chronic vitritis and 17 patients (23 specimens) with IOL were included. The specificity of vitreous fluid cytopathology, flow cytometry, and AIGHR was 1.0, and the sensitivity values were 0.24, 0.36, and 0.64, respectively. AIGHR was negative in two patients for whom cytopathology or flow cytometry revealed the diagnosis of lymphoma. Clonal IGHR was found in four specimens classified as negative for lymphoma based on cytopathology and flow cytometry. AIGHR supplemented cytopathology and flow cytometry to increase the diagnostic yield in IOL.

Eradication of Minimal Residual Disease during Treatment of Mantle Cell Lymphoma: CALGB 59909.

Recent studies suggest that detection of subclinical, or minimal residual disease (MRD) in apheresis products used for autografting correlates with poor disease-free survival following ASCT for mantle cell lymphoma (MCL). To validate this observation and to gain insights into the kinetics of MRD during treatment, we are performing a prospective analysis of MRD using quantitative real-time PCR (Q-PCR) in patients (pts) undergoing treatment for MCL on a CALGB study (59909). Q-PCR of sequential paired bone marrow (BM) and blood (B) samples and of apheresis products was performed using either a patient-specific immunoglobulin heavy chain (IgH) or BCL-1 gene rearrangement. All samples were analyzed in triplicate using LightCycler technology and reported as a normalized ratio of IgH or BCL-1 copy number to GAPDH copy number. The sensitivity of the assay ranged from 1 X 104–1 X 105. To date, a clonal IgH or BCL-1 gene rearrangement was detected in 36 of 41 (88%) pts entered on study. Patient-specific primers and consensus probes were used for Q-PCR monitoring of MRD following two courses of intensive induction therapy, during stem cell mobilization, and 3 and 12 months after ASCT and post-transplant immunotherapy with Rituximab (R). 27 pts have completed all protocol treatment with a median follow-up of 7 months (range: 0–28). 26 pts were evaluated for MRD following two courses of induction therapy with cyclophosphamide, methotrexate, doxorubicin, vincristine, prednisone, and R. 10 of 26 became MRD negative (−) following induction while 16 remained MRD positive (+). Following mobilization with high-dose cytarabine, etoposide and R, apheresis products from 9 of 10 MRD- pts were evaluated and all products were MRD- (1 pt not evaluable). Of the 16 pts who were MRD+ prior to mobilization therapy, MRD- apheresis products were collected in 8; 5 had MRD+ stem cell collections, and 3 were not evaluable. In total, apheresis products were evaluable in 22 of 26 pts; 17 (77%) had MRD- stem cells collected prior to ASCT. None of these MRD- pts has relapsed to date although 2 pts with MRD- products became weakly MRD+ 12 months following ASCT and R. Of the 5 pts with MRD+ apheresis collections, 4 have remained persistently MRD+ following ASCT and R; 1 has relapsed 19 months after completion of all treatment. Rising levels of MRD in BM and B samples were noted in this patient 3 and 12 months post-ASCT. Statistical comparison of MRD values in paired BM and B samples prior to, and post-ASCT demonstrated good agreement with an intraclass correlation coefficient of.814 and.777, respectively. In conclusion, our results demonstrate that prospective MRD monitoring using Q-PCR provides important insights into the kinetics of response during treatment of MCL. MRD- apheresis products were collected in the majority of pts following intensive induction and mobilization chemo-immunotherapy with R on CALGB 59909. To date, no pts with MRD- apheresis products have relapsed following ASCT. In contrast, it appears that pts with MRD+ apheresis products remain MRD+ following ASCT and R and may be more likely to relapse. Longer clinical follow-up is needed to clarify the significance of the persistence of MRD in apheresis products and following ASCT for MCL.