IgH Fusion Research Articles

Plasmablastic lymphoma with MYC::IGH fusion and BCL2 rearrangement.

A 50-year-old male with history of nodal stage III diffuse large B-cell lymphoma (DLBCL), refractory to R-CHOP, presented with vomiting, abdominal pain, and an umbilical mass. Abdominal computed tomography (CT) scan showed bulky retroperitoneal adenopathy and possible bowel obstruction. Histologic examination of the umbilical mass needle biopsy demonstrated diffuse atypical cells with medium to large nuclei, prominent central nucleoli, and eccentrically placed amphophilic cytoplasm (Figure 1, top left panel, objective 100×). The original lymphoma was of germinal center B-cell phenotype, was MYC-negative by immunohistochemistry (IHC), and had BCL2 rearrangement (BCL2-R) without MYC-R by interphase fluorescence in situ hybridization (FISH). In contrast, the umbilical mass neoplastic cells expressed plasma cell markers CD138 (Figure 1, top right, objective 60×) and MUM1 (Figure 1. Bottom left, objective 60×) and MYC (90%), were lambda light chain restricted, had a very high Ki-67 proliferation index (>90%), and were negative for all B-cell markers, germinal center cell markers, and Epstein-Barr virus. Interphase FISH studies identified BCL2-R and IGH::MYC fusion (Figure 1, bottom right, Ch 8 centromere: aqua probe, MYC [8q24]: red probe, IGH [14q32]: green probe; yellow fusion indicates IGH::MYC) without BCL6-R. These findings are characteristic of plasmablastic lymphoma with MYC::IGH fusion and BCL2-R. This likely represents transformation of the patient's prior DLBCL, with clonal evolution characterized by acquisition of MYC-R. Transformation of follicular lymphoma to plasmablastic lymphoma with MYC-R and BCL2-R has been previously described, but to our knowledge this is the first reported case of transformation of DLBCL to plasmablastic lymphoma with MYC::IGH fusion and BCL2-R. The “double-hit” cytogenetic features may portend an aggressive clinical course, similar to what has been observed in “high-grade B-cell lymphomas with MYC-R and BCL2-R and/or BCL6-R” (so-called double-hit lymphoma). The authors would like to thank the Mayo Clinic Genomics Laboratory for their continuous assistance and support of our service. The authors declare they have no conflict of interest. The authors received no specific funding for this work. Mohammad Barouqa wrote the manuscript. Patricia Greipp reviewed the article and cytogenetic probes. Rebecca King reviewed the article and stains. Ellen D. McPhail supervised the manuscript and finalized it.

Minimal Disease Monitoring in Pediatric Non-Hodgkin’s Lymphoma: Current Clinical Application and Future Challenges

Simple SummaryMinimal residual disease (MRD) describes the detection of a few remaining malignant cells in blood or bone marrow with molecular methods or flow cytometry. The detection of MRD in patients with leukemia during therapy indicates the risk of relapse. Its use for treatment stratification is state of the art in all modern leukemia treatment protocols. In pediatric non-Hodgkin lymphoma patients, minimal amounts of tumor cells, so-called minimal disseminated disease (MDD), can often be detected in blood or bone marrow at diagnosis. In children with ALK-positive anaplastic large cell lymphoma, MDD and MRD detected in the blood or bone marrow is associated with high relapse risk. For patients with Burkitt lymphoma or -leukemia or lymphoblastic lymphoma, the meaning of MDD and MRD is less clear. This review summarizes the current knowledge, techniques, application, and challenges in minimal disease detection in pediatric non-Hodgkin lymphoma.Minimal residual disease (MRD) detection is established routine practice for treatment stratification in leukemia and used for treatment optimization in adult lymphomas. Minimal disease studies in childhood non-Hodgkin lymphomas are challenged by stratified treatment in different subtypes, high cure rates, low patient numbers, limited initial tumor material, and early progression. Current clinical applications differ between the subtypes. A prognostic value of minimal disseminated disease (MDD) could not yet be clearly established for lymphoblastic lymphoma using flow cytometry and PCR-based methods for T-cell receptor (TCR) or immunoglobulin (IG) rearrangements. MYC–IGH fusion sequences or IG rearrangements enable minimal disease detection in Burkitt lymphoma and -leukemia. An additional prognostic value of MDD in Burkitt lymphoma and early MRD in Burkitt leukemia is implicated by single studies with risk-adapted therapy. MDD and MRD determined by PCR for ALK-fusion transcripts are independent prognostic parameters for patients with ALK-positive anaplastic large cell lymphoma (ALCL). They are introduced in routine clinical practice and used for patient stratification in clinical studies. Early MRD might serve as an endpoint for clinical trials and for guiding individual therapy. Validation of MDD and MRD as prognostic parameters is required for all subtypes but ALCL. Next-generation sequencing-based methods may provide new options and applications for minimal disease evaluation in childhood lymphomas.

29. Nanomapping with Cas9: A new high-resolution and efficient method for characterizing chromosomal rearrangements

Improvements in the genomic tools available for detecting chromosomal rearrangements (CREA) have resulted in the recognition of many new pathognomonic CREA/cancer associations, with these discoveries providing a basis for the development of new treatment modalities and prognostic stratifications. Excitement regarding the application of this CREA knowledge for patient management is accompanied by the recognition of a growing need for an improved assay that can yield a precise, high-resolution characterization of these CREA in a manner that is time-, cost- and specimen-use-efficient. To address this need, we developed “nanomapping”: a technique in which we (1) develop a unique “barcode” for a region of interest using nuclease-inhibited Cas9 molecules as stable and specific programmable labels; and (2) measure the lengths of DNA fragments and distances between Cas9 landmarks with nanoscale accuracy using high-speed atomic force microscopy. By comparing the barcode to a reference sequence, one can identify alterations in “long read” (≥100kb) DNA molecules at high speed and low cost ($1 per sample). To adapt this technique for CREA characterization, we: (1) used inverse PCR to amplify a translocation site of interest with multiplexed endonucleases to yield a characteristic profile of amplicon lengths; (2) nanomapped the resulting amplicons; and (3) compared the nanomaps to predicted reference maps generated in silico to obtain precise (high accuracy/specificity) identification of the chromosomal origin/breakpoints of fusion partners. As “proof of principle” for the utility of this new assay, to date, we have correctly identified IGH fusion partners in 4 neoplasms (including IGH/BCL2 translocations in 3/3 specimens and an IGH/MAFB translocation in 1/1 specimen). In summary, our data demonstrates the feasibility of using nanomapping to characterize CREA that have recurrent breakpoints. Moreover, it not only provides a means for enhancing our efficiency/accuracy in known CREA identification, but has potential to allow for the recognition of novel CREA.

Molecular profiling of Chinese R-CHOP treated DLBCL patients: Identifying a high-risk subgroup.

Diffuse large B-cell lymphoma (DLBCL) is a clinically aggressive and heterogenous disease. Although most patients can be cured by immunochemotherapy, 30% to 40% patient will ultimately develop relapsed or refractory disease. Here, we investigated the molecular landscapes of patients with diverse responses to R-CHOP. We performed capture-based targeted sequencing on baseline samples of 105 DLBCL patients using a panel consisting of 112 lymphoma-related genes. Subsequently, 81 treatment-naïve patients with measurable disease and followed for over 1 year were included for survival analysis. Collectively, the most commonly seen mutations included IGH fusion (69%), PIM1(33%), MYD88 (29%), BCL2 (29%), TP53 (29%), CD79B (25%) and KMT2D (24%). Patients with TP53 mutations were more likely to have primary refractory disease (87.0% vs 50.0%, P = .009). For those with TP53 disruptive mutations, 91.7% patients were in the primary refractory group. Interestingly, BCL-2 somatic hypermutation was only seen in patients without primary refractory disease (P = .014). In multivariate analysis, BCL-2 amplification (hazard ratio [HR] = 2.94, P = .022), B2M mutation (HR = 2.99, P = .017) and TP53 mutation (HR = 3.19, P < .001) were independently associated with shorter time to progression (TTP). Furthermore, TP53 mutations was correlated with worse overall survival (P = .049). Next, we investigated mutation landscape in patients with wild-type (WT) TP53 (n = 58) and found that patients harboring MYD88 L265P had significantly inferior TTP than those with WT or non-265P (P = .046). Our study reveals the mutation spectrum of treatment-naive Chinese DLBCL patients. It also confirms the clinical significance of TP53 mutations and indicates the prognostic value of MYD88 L265P in TP53 WT patients.

HnRNP L-dependent protection of normal mRNAs from NMD subverts quality control in B cell lymphoma.

The human nonsense-mediated mRNA decay pathway (NMD) performs quality control and regulatory functions within complex post-transcriptional regulatory networks. In addition to degradation-promoting factors, efficient and accurate detection of NMD substrates involves proteins that safeguard normal mRNAs. Here, we identify hnRNP L as a factor that protects mRNAs with NMD-inducing features including long 3'UTRs. Using biochemical and transcriptome-wide approaches, we provide evidence that the susceptibility of a given transcript to NMD can be modulated by its 3'UTR length and ability to recruit hnRNP L. Integrating these findings with the previously defined role of polypyrimidine tract binding protein 1 in NMD evasion enables enhanced prediction of transcript susceptibility to NMD. Unexpectedly, this system is subverted in B cell lymphomas harboring translocations that produce BCL2:IGH fusion mRNAs. CRISPR/Cas9 deletion of hnRNP L binding sites near the BCL2 stop codon reduces expression of the fusion mRNAs and induces apoptosis. Together, our data indicate that protection by hnRNP L overrides the presence of multiple 3'UTR introns, allowing these aberrant mRNAs to evade NMD and promoting BCL2 overexpression and neoplasia.

RNA Sequencing of Newly Diagnosed Multiple Myeloma to Identify a Predictive Signature for Proteasome Inhibitor-Based Therapy

A major challenge in modern myeloma management is the selection of the best initial treatment from a plethora of available therapies. PAD (bortezomib, doxorubicin, and dexamethasone) is a highly effective regimen which may obviate the need for an upfront high-dose melphalan stem cell transplant (HDSCT). However, it is not clear a priori which patients will benefit from this treatment. In the phase 2 PADIMAC trial, patients with newly diagnosed myeloma were treated with six cycles of PAD and stratified to receive HDSCT in partial remission (PR) or watch and wait in very good partial remission (VGPR) or better. To identify predictive biomarkers, we extracted somatic RNA prior to treatment and performed massively parallel RNA sequencing (RNAseq).Basic quality control metrics suggested a high-quality RNAseq dataset. Furthermore, spiked expression of IgH fusion partners was consistent with fluorescent in situ hybridization (FISH) data for each sample. All t(4;14) and t(4;16) cases were correctly identified by RNAseq on the basis of the relevant fusion transcript. As a final quality control measure, recurrent variants (e.g. NRAS, KRAS, TP53, FAM46C, DIS3) were identified in the expected proportions.We performed a pre-planned analysis of differential gene expression between those patients achieving a VGPR or better, sustained for at least 12 months without HDSCT (excellent responders), and those patients who achieved less than a PR (poor responders). 85 genes were upregulated in poor responders and 225 in excellent responders. Interestingly, there was no significant overlap between our PAD-predictive signature and the Arkansas 70 gene poor prognosis signature, the Arkansas 17 gene poor prognosis signature, or the EMC92 prognostic signature. This is likely to reflect in part, the greater sensitivity of RNAseq for gene expression compared to microarrays. However, it also implies that there is a selective signature for PAD responsiveness.We employed the Gage and Pathview packages to identify non-redundant pathways associated with PAD responsiveness. Significantly upregulated genes in poor responders were those involved in: DNA replication, base excision repair, and the Fanconi anaemia pathway. Genes upregulated in excellent responders included those involved in: several signalling pathways (including RAS, NF-Kappa B, FOXO and JAK-STAT pathways); chemokine and cell adhesion; protein processing in the endoplasmic reticulum; and immune activation pathways such as T-cell receptor signalling and natural killer cell-mediated cytotoxicity.Finally, we investigated the ability of expression signatures to stratify patients on the basis of PAD responsiveness. Our analysis suggests that RNASeq-defined transcriptional profiles could aid in the selection of patients for PAD therapy (leading to an increase in overall response rate from 70% to 86%), as well as the stratification of patients in CR to a non-HDSCT protocol. We anticipate that our current analysis could easily incorporate specific response signatures for other regimens and could make personalized therapy for myeloma a reality in the foreseeable future. DisclosuresYong:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based chimeric antigen receptor; Janssen: Research Funding. Streetly:Guys and St. Thomas’ NHS Trust: Honoraria. Schey:Celgene, Takeda: Honoraria; Celgene: Consultancy; Celgene, Johnson & Johnson: Speakers Bureau. Cook:Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Glycomimetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria. Cook:Takeda: Honoraria; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Myeloma UK: Membership on an entity’s Board of Directors or advisory committees.

The Fusion Gene Landscape in Multiple Myeloma, with Clinical Impact

Background: Gene fusions play an important role in aberrant cellular biology as well as development and progression of cancer. Expression of fusion genes such as PML-RAR drives the transformation in APL and provides important targets for therapy. However, in multiple Myeloma (MM), a heterogeneous disease characterized by genomic instability, frequent gains and losses of DNA, and a diverse mutational landscape, only the well characterized MMSET-IGH fusion product has been reported. Here we investigate the fusion gene landscape in multiple myeloma, and its possible impact on survival.Method: Deep RNA-Seq was performed on purified MM cells from 430 newly-diagnosed MM patients, 20 normal individuals and 71 cell-lines; data were analyzed for gene expression profiles, long-non coding RNA signatures, and both novel and known fusion genes using two common algorithms: TopHat and MapSplice. MM characteristics, cytogenetic and FISH as well as clinical survival outcomes were also analyzed and correlated with genomic data.Results: After filtering candidate fusions linking genes belonging to the same family, we identified 416 different candidates in myeloma patients, 40 % of which identified either IGH or Kappa as a partner. IGH fusion partners included the previously described and validated WHSC1 and B2M genes, as well as over 50 new candidates, while more than 70 different partners were found to be fused with Kappa. These genes exhibit functional enrichment of positive regulators of the cytokine-mediated signaling pathway, negative regulation of myeloid cell differentiation, negative regulation of interleukin-6 production, as well as others. 31% of patients presented no fusions, and another 32% presented a single fusion event. The other 37% presented at least 2 fusion candidates, with up to 27 different candidates. Similar patterns were observed in cell-lines, with 196 unique candidates identified, only 16% of which involving IGH or kappa. However, all partners were found in at least one patient as well. Only 12% of cell-lines exhibited no fusion, and another 14% presented only one fusion. On average, 3 fusions were identified per cell-line, with a maximum of 10. Validation of some these fusion genes is required to understand their functional role. Importantly, although having IgH-or kappa-related fusions did not affect patient outcome by themselves, patients with high numbers of fusion candidates had worse event-free survival.Conclusion: Our data describes a diverse and rich fusion gene landscape in Multiple Myeloma. Similar to mutational profiles, there is no predominant fusion gene driving the disease process. Association of poor prognosis with a higher number of fusions may indicate that genomic instability plays an important role in the biology of Multiple Myeloma. DisclosuresNo relevant conflicts of interest to declare.

Frequent Igh Fusion Transcripts with Clinical Impact in Multiple Myeloma

Background: Significant heterogeneity has been described in Multiple Myloma (MM), especially at the genomic level. Frequent gains and losses of DNA along with various mutations have been observed, and differential allelic expression is being characterized. Fusion proteins are common and maybe associated with cell transformation, growth and lethality of tumor cells. However, unlike in leukemia, no consistent fusion gene product has been consistently identified. As IgH-related translocations have an important role in myeloma, we have investigated fusion genes involving IgH, to understand their biology and explore a possible effect on survival in MM.Methods: We performed deep RNA-Seq on purified MM cells from 430 newly-diagnosed MM patients and analyzed gene expression profiles, isoform signatures and both novel and known fusion genes using two common algorithms: TopHat and MapSplice. We also correlated genomic data with patient data including cytogenetics and FISH, as well as survival.Results: We primarily focused on fusions involving the IGH gene (chromosome 14) and found that about one fourth of the patients (57 out of 430) presented an IGH fusion gene (97 patients according to TopHat, 303 according to Mapsplice, 57 according to both). These included the well described t(4,14) fusion involving the MMSET gene (found in 47 patients by both algorithms, 49 by Tophat, 54 by Mapsplice). Additionally we observed fusions involving chromosomes 14 and chromosomes 1, 4, 11, 12, and 16. The counterpart genes involved in the IgH fusions included PDE3A (chromosome 12 - 4 patients); HFM1 (chromosome 1, 2 patients); NFKB1, FGFR3, CIITA, WWOX and MRPL21 (chromosomes 4, 16, and 11, 1 patient). As RNA-Seq data allows the precise localization of the breakpoints, we were able to identify that out of the 47 t(4,14) patients, 62% were MB4-1, 9.5% were MB4-2 and 28.5% were MB4-3. Interestingly, we did not see fusion products involving IgH and other known parts on Chromosomes 8 and 20. We studied event-free survival in a subset of 265 patients with available survival data and found that, as predicted, patients with an IgH-MMSET fusion had significantly lower survival than others. However, patients with a fusion gene involving IGH and any other partner have a significantly better prognosis as a group. Moreover, the poor prognosis of IgH-MMSET fusion appears to be driven by MB4-3 patients. Importantly, the fusions identified using RNA-seq were also validated by FISH analysis. All t(4,14) fusions were characterized by a very high MMSET expression (FPKM greater than 20) while patients with other fusions presented a lower MMSET expression (FPKM lower than 10).Conclusion: Our study suggests that IgH-related translocations in myeloma may impact tumor biology by a number of mechanism, one of which is the generation of fusion proteins with functional consequences. It also highlights a possible clinical impact that requires validation in larger cohorts. DisclosuresNo relevant conflicts of interest to declare.

Bcl-2/IgH Expression of Diffuse Large B-cell Lymphoma Cells in Minimal Bone Marrow Infiltration

We sought to investigate the role of the Bcl-2 translocation at the chromosomal and protein levels in minimal bone marrow (BM) infiltration by diffuse large B-cell lymphomas (DLBCL). The presence of the Bcl-2/IgH fusion gene was detected in BM samples and paraffin-embedded lymph node (LN) samples from 103 patients with DLBCLs using FISH. Bcl-2 protein levels in BM and paraffin-embedded tissues were quantified using immunocytochemistry (ICC) and immunohistochemistry (IHC), respectively. Bcl-2/IgH translocation in paraffin-embedded LN tissue sections was observed in 43 (41.7%) patients by FISH. Of the 43 patients, the Bcl-2/IgH rearrangement in the bone marrow specimens occurred in 34 patients. The Bcl-2/IgH recombination rate in stage III cancers was not significantly different compared to the rate observed in stage I/II cancers (p = 0.101), respectively showing no statistical differences between stage IV and I/II (p = 0.179). In 64.7% (22/34) of the cases with t(14;18), Bcl-2 was detected based on ICC analysis. Positive Bcl-2 ICC staining and the t(14;18) translocation were positively correlated (p < 0.001). We then applied our FISH method to slides with at least one abnormal cell and were subjected to FISH analysis after staining. During the follow-up, no infiltration by cytomorphology for 16 DLBCL patients whose bone marrow presented Bcl-2/IgH gene rearrangement at diagnosis, and two cases were positive by morphology compared to FISH-positive results 6 months later; 9 out of 16 patients (56.3%) presented with positive Bcl-2/IgH results earlier than the morphology evaluation after 12 months. Utilizing both FISH and cytologic morphology, the assessment of Bcl-2/IgH translocation status could contribute to the better detection of minimal bone marrow infiltration and relapse receiving treatment by DLBCL cells.

Bcl-2/IgH expression in minimal bone marrow infiltration by follicular lymphoma cells

The purpose of this study was to investigate the roles of bcl-2 chromosomal translocation and Bcl-2 protein expression in follicular lymphoma (FL) minimal bone marrow (BM) infiltration. We identified the same bcl-2/IgH fusion gene in paraffin-embedded lymph node (LN) samples and BM samples using immunohistochemistry (IHC), immunocytochemistry (ICC), cytologic morphology and fluorescence in situ hybridization (FISH). The presence of the Bcl-2/IgH fusion gene in the BM samples and paraffin-embedded LN samples from 56 patients with follicular lymphomas was detected using FISH. The Bcl-2 protein levels in BM and paraffin-embedded tissues were quantified using ICC and IHC, respectively. Approximately 78.6% (44/56) of the paraffin‑embedded LN tissue sections that underwent FISH analysis had a bcl-2/IgH translocation. The primary lesion was also positive for the bcl-2/IgH fusion gene, as were the BM minimal infiltrates. The bcl-2/IgH rearrangement occurred in 88.6% (39/44) of the BM specimens. The bcl-2/IgH recombination rate in stage III/IV cancers was significantly different to that observed in stage I/II cancers (p=0.041). In 59% (23/39) of the cases with t(14;18), Bcl-2 was found to be present as assessed by ICC. Positive Bcl-2 ICC staining and the t(14;18) translocation (as detected using FISH) were positively correlated (p=0.028). We then applied the FISH method to slides that had previously been morphologically evaluated using Wright-Giemsa staining; any slides with at least one abnormal cell were subjected to FISH analysis following staining. The assessment of bcl-2/IgH translocation status may contribute to the better detection of minimal BM infiltration by FL cells. Utilizing FISH and cytologic morphology techniques allows for earlier and more accessible BM examination.

Identification of Bcl-2/IgH fusion sequences using real-time PCR and chip-based microcapillary electrophoresis

The determination of polymerase chain reaction (PCR) amplification product sizes of the Bcl-2/IgH fusion gene from follicular lymphoma (FL) provides evidence of clonal identity. The present study describes detection of Bcl-2/IgH fusion gene clonality utilizing a small, simple microcapillary electrophoretic chip combined with a real-time PCR method. The microcapillary electrophoretic chip system effectively detects size differences among the Bcl-2/IgH fusion gene amplification products of FL from patient samples; something that is not possible using traditional gel electrophoresis. We also describe the potential of this system to utilize formalin-fixed, paraffin-embedded tissue samples sectioned on charged slides. The simple detection of Bcl-2/IgH fusion gene clonality using a microcapillary electrophoretic chip provides reliable information for monitoring minimal residual disease of FL, and can be an effective tool for use in clinical laboratories.

ζ‐Crystallin is a bcl‐2 mRNA binding protein involved inbcl‐2overexpression in T‐cell acute lymphocytic leukemia

The human antiapoptotic bcl-2 gene has been discovered in t(14;18) B-cell leukemias/lymphomas because of its overexpression caused at a transcriptional control level by the bcl-2/IgH fusion gene. We were the first to disclose the post-transcriptional control of bcl-2 expression mediated by interactions of an adenine + uracil (AU)-rich element (ARE) in the 3'-UTR of bcl-2 mRNA with AU-binding proteins (AUBPs). Here, we identify and characterize zeta-crystallin as a new bcl-2 AUBP, whose silencing or overexpression has impact on bcl-2 mRNA stability. An increased Bcl-2 level observed in normal phytohemagglutinin (PHA)-activated T lymphocytes, acute lymphatic leukemia (ALL) T-cell lines, and T cells of patients with leukemia in comparison with normal non-PHA-activated T lymphocytes was concomitant with an increase in zeta-crystallin level. The specific association of zeta-crystallin with the bcl-2 ARE was significantly enhanced in T cells of patients with ALL, which accounts for the higher stability of bcl-2 mRNA and suggests a possible contribution of zeta-crystallin to bcl-2 overexpression occurring in this leukemia.

In situ localization of follicular lymphoma: evidence for subclinical systemic disease with detection of an identical BCL-2/IGH fusion gene in blood and lymph node

In situ localization of follicular lymphoma: evidence for subclinical systemic disease with detection of an identical BCL-2/IGH fusion gene in blood and lymph node

Chromosome Translocations in Workers Exposed to Benzene

As benzene has been linked with elevated risk of both acute myeloid leukemia and lymphoma, we explored the effect of benzene exposure on levels of t(8;21), t(15;17), and t(14;18) translocations. Circulating lymphocytes of normal individuals also often contain t(14;18). Quantitative polymerase chain reaction analysis showed that 37 workers with benzene exposure had a decreased level of t(14;18) in their blood with only 16.2% having 10 or more copies of the t(14;18) BCL-2/IgH fusion gene/microg DNA, as opposed to 55% of 20 controls (P = .0063 by Fisher's exact test). This decline may be related to the immunotoxicity to specific subtypes of circulating B-lymphocytes, but the data do not support the use of t(14;18) as a biomarker of increased lymphoma risk in benzene-exposed populations. None of 88 individuals (31 controls and 57 exposed) exhibited detectable t(8;21) transcripts, and while t(15;17) transcripts were detected in two individuals, the result is inconclusive as one was exposed and the other was unexposed.

Multiplex PCR for the detection of BCL-1/IGH and BCL-2/IGH gene rearrangements – clinical validation in a prospective study of blood and bone marrow in 258 patients with or suspected of non-Hodgkin's lymphoma.

We have designed a multiplex PCR, which allows for fast and high throughput demonstration of the BCL-1/IGH and BCL-2/IGH fusion DNA observed primarily in mantle cell- and follicular non-Hodgkin's lymphoma (NHL). Blood (PB) and/or bone marrow (BM) from 258 patients suspected of NHL have prospectively been evaluated. Eleven patients (4%) were found t(11;14)+ and 37 patients (14%) t(14;18)+. Comparing these results to standard diagnostic methods of PB and/or BM identified PCR+ samples that were normal by morphology (BCL-1/IGH: 1/11; BCL-2/IGH: 17/37). Equally important, patients who were not clonal in PB and/or BM by flow cytometry were identified as PCR+ (BCL-1/IGH: 3/11; BCL-2/IGH: 23/37). We conclude that this multiplex approach allows for easy and sensitive molecular determination of molecular lesions in NHL, which have diagnostic and prognostic importance.

Blastoid Variant of Mantle Cell Lymphoma with Lactic Acidosis: A Case Report

Approximately 20% of mantle cell lymphomas (MCL) present with the blastoid variant associated with poor prognosis. Lactic acidosis complicated with hematologic malignancies is seen infrequently and is associated with a poor outcome. Here we report the case of a patient with the blastoid variant of MCL complicated by lactic acidosis and who achieved complete remission through chemotherapy combined with rituximab therapy. A 77-year-old man presented with peripheral blood lymphoma cells, huge splenomegaly, abdominal and mediastinal lymphadenopathy, and pleural effusion. A bone marrow smear showed an increase in large, abnormal lymphoid cells with oval or round nuclei, distinct nucleoli, and abundant basophilic cytoplasm with vacuolization. Splenic sections also showed massive and diffuse infiltration by these cells. Flow cytometry analysis showed these cells to be positive for CD5, CD19, CD20, and kappa chain and negative for CD10 and CD23. A blastoid variant of MCL was diagnosed from the results of histologic, immunohistochemical (cyclin D1), and cytogenetic (chimeric bcl-1/IgH fusion gene) analyses. The patient recovered from the 2 episodes of severe lactic acidosis for which he had been given chemotherapy, and he achieved complete remission after subsequent chemotherapy combined with rituximab treatment.

Establishment and characterization of a new human myeloma cell line, KYδ-1, producing the δ/κ type immunoglobulin

We describe the establishment and characterization of a new multiple myeloma (MM) cell line, KYδ-1, which expressed δ/κ type immunoglobulin (Ig). The patient was a 65-year-old woman with MM, who presented extramedullary dissemination, lymphadenopathy and short survival. The KYδ-1 cell line was derived from the pleural fluid obtained in the terminal phase of the disease. The cells expressed δ/κ Ig in the cytoplasm, and CD10, CD29, CD33, CD38, CD44, CD54, and HLA-DR antigens on the cell surface. Chromosomal analysis revealed two independent translocations, t(3;14)(p21;q32) and t(3;11)(p21;q13), which were confirmed by fluorescence in situ hybridization using chromosome painting probes. Reverse transcriptase-mediated polymerase chain reaction (PCR) and Northern blot analyses demonstrated overexpression of the CCND1 gene, suggesting alteration of the BCL1- CCND1 locus. We thus performed long-distance inverse PCR using nested primers for the Cα constant region of immunoglobulin heavy chain gene ( IGH) and obtained a clone that encompassed the 11q13/ IGH fusion. Nucleotide sequencing determined that the fusion occurred at the Sα2 switch region and at the centromeric side of the major translocation cluster of BCL1. The other IGH allele consisted of a VDJ complex that was adjacent to the Cδ constant gene, indicating that a class switch-like mechanism from the Cμ to Cδ was involved in the production of the Ig δ heavy chain. Point mutations within the P53 and N-RAS genes were presumably related to the rapidly progressive disease in this particular MM patient.