Rearranged Immunoglobulin Heavy Chain Research Articles

Clinicopathologic features of primary thymic extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue type

To study the clinicopathologic features of primary thymic extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT). The clinical and pathologic findings were evaluated in 3 cases of biopsy confirmed thymic MALT lymphoma. The clincopathologic features, treatment and prognosis were discussed and literatures reviewed. One male and two female patients presented with asymptomatic mediastinal masses with a history of Sjögren syndrome. They were aged 36, 35 and 41 years respectively, and only one patient had B symptoms. Grossly, all three tumors were encapsulated and had multiple variable-sized cysts on cut-surface. Histopathologically, the normal thymic lobular architecture was effaced by abnormal dense lymphoid infiltration. Prominent lymphoepithelial lesions were formed by centrocyte-like cells infiltrating and expanding Hassall's corpuscles and epithelial cyst lining. All cases showed apparent plasmacytic differentiation. Immunohistochemically, the tumor cells were positive for CD20, CD79a, bcl-2 and negative for CD3, CD5, cyclin D1, CD43, CD10, bcl-6, and CD23. The plasma cells showed kappa light chain restriction. Immunoglobulin heavy chain rearrangement in three cases was confirmed by PCR. All patients were at early stage and received routine chemotherapy with or without radiotherapy after surgical removal. All patients achieved complete remission with 24, 18 and 3 months follow-up, respectively. Primary thymic MALT lymphoma may be a rare distinctive lymphoma. It can be diagnosed by HE and immunohistochemical study and should be differentiated from reactive lymphoid proliferation, other types of lymphoma and mediastinal thymoma.

Phenomenon of Oligoclonality in Adult Patients with Acute Lymphoblastic Leukemia

Abstract 4895 Introduction:Minimal residual disease (MRD) in adult patients with acute lymphoblastic leukemia (ALL) can be analyzed by flow cytometry, fluorescent in situ hybridization (FISH) if there are chromosomal abnormalities, polymerase chain reaction (PCR) for chimerical transcripts and immunoglobulin heavy chain (IgH) and T-cell receptor gamma (TCRG) genes rearrangements. IgH and TCRG rearrangements are the for monitoring of MRD, because it is possible to find specific clonal tumor marker in 80–90% patients. According to the data of different research groups phenomenon of oligoclonality (more than one clonal rearrangement) is identified in 16–80% cases depending on how much molecular markers are used (Scrideli CA, 2001; Gameiro P, 2002; Thorsten Raff, 2006; Csinady E, 2009; Katsibardi K, 2011). Methods and materials:We studied bone marrow samples from 59 patients with new diagnosed B-lineage ALL (43 patients), T-lineage ALL (15 patients) and one patient with bilinear B-T-ALL. Ph-positive ALL was diagnosed in 6 patients, t(4;11) – in one patient. Patients were treated by standard ALL-protocols based on 8 weeks induction remission, 3–5 consolidation courses and maintenance treatment during 2 years. Patients with Ph-positive B-ALL were treated by the same chemotherapy with imanitinib and one patient with mature B-ALL was treated by NHL-BFM-90 chemotherapy. Detection of IgH and TCRG genes rearrangements were carried out by polymerase chain reaction using standard family-specific primers. Synthesis of patient specific primers was based on defined nucleotide sequence of clonal PCR-product. Monitoring MRD was realized by nested PCR using patient specific primers. Results:IgH and/or TCRG genes rearrangements were detected in 50 of 59 patients (84,7%). IgH complete rearrangements were found in 31of 43 patients (72,1%) with B-lineage ALL, IgH incomplete rearrangements were detected in 12 of 43 patient (27,9%) with B-lineage ALL and in one of 15 patient with T-lineage ALL. TCRG rearrangements were found in 13 of 15 patients (86,7%) with T-lineage ALL and in 24 of 43 patients (55,8%) with B-lineage ALL. Seventeen (34%) of 50 patients had one clonal marker. So, phenomenon of oligoclonality was revealed in 33 patients (66%). Two clonal rearrangements were detected in 23 cases, 3 rearrangements were detected in 7 cases and 3 patients had 4 rearrangements. In 3 of 27 patients, in whom all markers were followed, monitoring of MRD showed different pattern of tumor subclones elimination. Monitoring of MRD in patients who had also chromosomal abnormalities such as t(9;22), t(4;11) was carried out by FISH and PCR in Real-time. In one case the patient had 3 IgH rearrangements and chromosomal abnormality t(4;11) at the diagnosis. Later at various stages of chemotherapy we observed a different clearance of IgH rearrangements, however, when cytogenetic remission was achieved and MLL-AF4 expression was absent there were two of three IgH markers. This fact confirms the presence of several tumor subclones, their different clearance during the treatment and suggests persistence of tumor in this patient. Conclusion:In ALL patients with several subclones MRD monitoring should carried out with all markers, as we proved different subclone clearance suggesting different cells sensitivity to cytostatic drugs. Disclosures:No relevant conflicts of interest to declare.

Molecular Analysis of RAS-RAF Tyrosine-Kinase Signaling Pathway Alterations in Multiple Myeloma

Abstract 2876Multiple myeloma (MM) is a malignancy of abnormal plasma cells and a correlation with poor outcome has been described for immunoglobulin heavy-chain (IgH) translocations, deletions of 13q or 17p. Thus far, a convincing relationship between specific mutations, disease onset and progression has not been fully established. Aberrant impairment of important signaling pathways can drive oncogenesis and contribute to MM development. We therefore chose to study NRAS, KRAS and BRAF, three members of the RAS-RAF signaling pathway, as well as TP53 and CCND1, two fundamental genes in cell cycle control. We here investigated 41 MM cases to further elucidate molecular mechanisms underlying this disease. Bone marrow (n=35) or, in case of plasma cell leukemia, peripheral blood (n=6) specimens were collected between 12/2006 and 6/2011 and molecular analyses using a deep-sequencing assay (454, Branford, CT) in combination with the 48.48 Access Array technology (Fluidigm, South San Francisco, CA) were performed on mononuclear cells after Ficoll enrichment or magnetic activated plasma cell sorting using anti-CD138 beads (RoboSep, STEMCELL Technologies SARL, France). The cohort included 16 female and 25 male patients at first diagnosis, with a median age of 63 years (range: 33–84 years). Based on fluorescence in situ hybridization (FISH), the cohort was characterized as follows: IgH rearrangements were detected in 54.3% of patients (19/35: n=6 with t(4;14), n=9 with t(11;14), n=3 with t(14;16), n=1 other; data not available: n=5). A deletion 13q14 was present in 64.9% of patients (24/37; data not available: n=4). Trisomy 3 was detected in 48.0% of patients (12/25; data not available: n=16), trisomy 9 was detected in 50.0% of patients (12/24; data not available: n=17), trisomy 11 was detected in 46.4% of patients (13/28; data not available: n=13), and trisomy 15 was detected in 56.2% of patients (9/16; data not available: n=25), respectively. Interestingly, in all cases where FISH data was available (n=36), at least 1 aberration was detectable. Further, we studied the occurrence of somatic mutations in NRAS, KRAS, BRAF, TP53 and CCND1. In our cohort, we detected an overall mutation rate within the RAS pathway of 41.4% (17/41), in line with a recent report (Chapman et al., Nature, 2011). KRAS was the most frequently mutated gene with 21.9% of cases with mutations (9/41 patients), followed by NRAS (19.5%; 8/41 patients). Recently, BRAF V600E mutations have gained clinical interest since they became manageable by targeted treatment in melanoma. Interestingly, Chapman et al. discovered a mutational rate of 4% by sequencing of 161 MM patients (Nature, 2011). Even if BRAF is not a frequently mutated gene in MM, it justifies upfront diagnostic screening since these patients may benefit from new treatments. In our cohort, 2/41 patients harbored BRAF V600E mutations. Moreover, because of their involvement in the same signaling pathway, we also noticed that mutations affecting NRAS, KRAS or BRAF were predominantly mutually exclusive, except for one patient who concomitantly harbored a BRAF and a NRAS mutation. Additionally, we observed an overall molecular TP53 mutation rate of 12.2% (5/41 patients). In these 5 patients, in total 7 mutations (5 missense substitutions; 2 frame-shift mutations) were detected. 1/4 cases concomitantly harbored a deletion of the TP53 gene, as detected by FISH. Finally, we were interested in the analysis of CCND1, which is located on 11q13, a region frequently involved in chromosomal translocations (9/20 IgH translocated cases in our cohort). Here, we were able to detect 2/41 (4.8%) CCND1 mutated cases. Concerning the correlation between IgH rearrangements and molecular aberrations we observed that 21.9% (9/35; n=5 IgH status not available) of patients that were IgH rearranged, concomitantly carried a TP53 or RAS-RAF mutation. In more detail, 2/5 TP53 mutated patients and 50.0% (8/16) RAS-RAF mutated cases concomitantly harbored an IgH rearrangement. Taken together, MM patients are currently stratified in part based on cytogenetic/FISH classification. We demonstrated that deep-sequencing analyses support an additional molecular characterization. In our cohort, all patients carried mutations detected by FISH and 23/41 (56.1%) carried a molecular mutation. Future clinical studies need to confirm the frequencies of these mutations as well as their association with response to therapy and outcome. Disclosures:Artusi:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment.

LONG-TERM RESULTS of the GIMEMA VTD Consolidation TRIAL In Autografted MULTIPLE Myeloma PATIENTS (VEL-03-096): IMPACT of Minimal RESIDUAL DISEASE Detection by REAL Time Quantitative PCR On LATE Recurrences and Overall SURVIVAL

Abstract 827 Background and aims:We have recently shown that a consolidation therapy with bortezomib/thalidomide/dexamethasone (VTD) in multiple myeloma (MM) patients responding to autologous transplantation (ASCT) induces major tumor shrinking assessed by real time-quantitative (RQ)-PCR. Moreover we found that low levels of minimal residual disease (MRD) associated to a better progression-free survival (PFS) [GIMEMA VEL-03-096 trial, EudraCT Number 2004-000531-28: Ladetto et al, J Clin Oncol 2010]. We here present the updated results of this study at a median follow-up of 65 months. In the present analysis the following additional issues have been addressed: a) impact of MRD on PFS over time, with special interest to the role of MRD kinetics on outcome; b) impact of MRD on overall survival (OS). Patients and methods:Inclusion criteria and treatment schedule for this study have been already reported [Ladetto et al., J Clin Oncol 2010] and included: 1) a documented complete or very good partial remission following ASCT delivered as first line treatment; 2) no previous therapy with thalidomide or bortezomib; 3) presence of a molecular marker based on the immunoglobulin heavy chain rearrangement (IGH). MRD was assessed on bone marrow samples at diagnosis, study entry, after two VTD courses, at the end of treatment and then at six months intervals, up to clinical relapse. Patients underwent MRD detection using either qualitative nested PCR and RQ-PCR, employing IGH-derived patient specific primers as already described [Voena et al., Leukemia 1997; Ladetto et al., Biol Bone Marrow Transpl 2000]. For outcome analysis patients were grouped according to following definitions: a) MRD negativity on two consecutive samples by the most sensitive PCR method (nested PCR): full molecular remission (FMR); b) MRD negativity on two consecutive samples by RQ-PCR (less sensitive but currently better standardized, according to European Study Group on MRD detection guidelines [van der Vendel et al., Leukemia 2007]): standard molecular remission (SMR); c) post-treatment tumor load above the median by RQ-PCR: high tumor burden (HTB); d) post-treatment tumor load below the median by RQ-PCR: low tumor burden (LTB); e) recurrence of detectable MRD after FMR/SMR: molecular relapse (M-rel); f) increase of MRD levels of at least one log: active disease (AD). Results:Feasibility, toxicity and clinical outcome of the trial have been already reported [Ladetto et al., J Clin Oncol 2010]. Thirty-nine patients were enrolled and median clinical follow-up from start of first line treatment is 65 months. 270 of the planned samples for MRD monitoring (86%) were actually received by the centralized lab. So far 17 relapses and six deaths have been reported. Following VTD consolidation, 7/38 evaluable patients achieved FMR (18%) and 15/38 achieved SMR (39%). Three M-rel were observed, two of them followed by clinical relapse within six months. Achievement of SMR proved highly predictive for PFS (5-years (y) PFS 82% vs 44%, p=0.009, figure 1A), as well as the presence of HTB and AD (5-y PFS 35% vs 87%, p<0.001, figure 2). Interestingly, patients with LTB and no evidence of M-rel or AD had an excellent outcome with a 5-y PFS of 87%, (even considering that molecular follow-up was incomplete due to lack of samples in the two events observed in the low risk group, figure 2). Most notably, none of the patients achieving FMR or SMR has so far died and both SMR and AD proved to be significant predictors for OS (respectively, 5y-OS 100% vs 74%, p=0.012, figure 1B, and 5y-OS 86% vs 100%, p=0.037, data not shown). [Display omitted] [Display omitted] Conclusions:Our long-term results indicate that: 1) the achievement of SMR following VTD consolidation in MM patients is associated with a better outcome in terms of PFS and OS; 2) a dynamic increase in molecular tumor burden (AD), detectable by RQ-PCR, predicts late disease relapses several months before clinical recurrence. Taken together these results suggest the importance of developing tailored treatment for patients with high residual burden or showing increasing levels of MRD during follow-up, as already pursued for example in mantle cell lymphoma [Andersen et al., J Clin Oncol 2009]. Disclosures:Ladetto:Celgene: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Bayer: Honoraria; Mundipharma: Honoraria; Janssen-Cilag: Research Funding; Italfarmaco: Research Funding. Cavallo:celgene: Honoraria. Guglielmelli:celgene: Honoraria; Janssen-Cilag: Honoraria. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Palumbo:Merck: Honoraria; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.

Tyrosine Kinase Inhibitor Therapy Induced Changes in Humoral Immunity in Patients with Chronic Myeloid Leukemia

Abstract 1699 Background:By affecting not only BCR-ABL1 but also a wide variety of other tyrosine kinases, tyrosine kinase inhibitors (TKIs) have potential impact on the immune defense system. As patients could use TKIs even for decades, immunological off-target effects may be of significance in clinical practice. The aim of this project was to study the effects of TKI therapy on B-cell-mediated immunity in vivo in CML patients. PATIENTS AND METHODS:Peripheral blood (PB) and bone marrow (BM) samples were collected from newly diagnosed chronic phase CML patients and follow-up samples were obtained during TKI therapy at 1, 3, 6, and 12 months after therapy start (imatinib n=21, dasatinib n=15 and nilotinib n=7). Samples from healthy volunteers served as controls (n=10). Plasma immunoglobulin (Ig) levels were measured with an immunoturbidometric method and BM and PB lymphocyte subclasses were analyzed with multicolor flow cytometry. Ig heavy chain rearrangement PCR according to BIOMED-2 protocol was used to identify clonal B-cell populations among sorted CD19pos B lymphocytes. RESULTS:At diagnosis, before TKI therapy, plasma Ig levels were similar to healthy controls. During imatinib treatment IgA and IgG levels decreased continuously and were significantly lower at 12 months compared either to diagnostic samples or to healthy controls: median IgA level at 12 months 1.22 vs. 1.51 g/l at diagnosis (p=0.040) or vs. 1.61 g/L in healthy controls (p=0.023); median IgG at 12 months 7.00 vs. 8.80 g/l at diagnosis (p=0.009) or vs. 8.55 g/l in healthy controls (p=0.009). Similarly plasma IgM levels decreased: 0.59 g/ at 12 months vs. 1.06 g/l at diagnosis (p=0.0002), but did not differ significantly from healthy controls (0.75 g/l, p=0.331). Positive correlation was found between plasma IgG and IgM levels at 3 months (r=0.678, p=0.045). One imatinib patient developed severe constant hypogammaglobulinemia, which was first noticed at 3 months, and imatinib was discontinued at 15 months due to recurrent respiratory infections.In dasatinib-treated patients, only plasma IgM levels decreased notably, and at 12 months, they were significantly lower (0.39 g/l) compared to diagnosis (0.86 g/l, p=0.011) or healthy controls (0.75 g/l, p=0.037).No statistically significant changes were observed in nilotinib-treated patients, possibly due to small number of patients.The CD45 expression analyzed by flow cytometry was used to detect different B-cell subclasses (CD45high as mature B-cells, CD45intermediate as maturating B-cells and CD45low as immature B-cells). In imatinib treated patients the proportion of mature B-cells in BM from all CD19+ cells was significantly lower at 3 months than in dasatinib group (55% vs. 84%, p<0.05), whereas the proportion of maturating B-cells was higher (34% vs. 12%, p<0.05). This could be related to the observed low plasma Ig levels in imatinib patients, as only mature B-cells are capable of secreting immunoglobulins. BM B-cell profile of nilotinib patients resembled that of imatinib patients.Although clonal T-cells are found in the majority of CML patients (Kreutzman et al. Blood 2010), Ig heavy chain rearrangement analysis did not reveal any clonal B-cell populations in CML patients at diagnosis (n=2) or during imatinib (n=2) or dasatinib (n=9) treatment. Similarly, no marked abnormalities were observed in serum electrophoresis or immunofixation analysis. CONCLUSIONS:Imatinib-treated patients have reduced plasma Ig levels and the proportion of mature immunoglobulin-secreting B-cells was lower in BM. Unexpectedly, dasatinib-induced changes were much less prominent, although dasatinib inhibits a broader spectrum of kinases, some of which are clearly involved in B lymphocyte biology, such as the Lyn-kinase. Further studies are needed to understand which kinase inhibition plays the major role in imatinib-induced reduction of Ig levels. However, the measurement of plasma Ig levels is warranted in patients with recurrent respiratory infections and possibility of hypogammaglobulinemia needs to be taken into account. Disclosures:Koskela:Novartis: Honoraria. Richter:Bristol-Myers Squibb: Honoraria; Novartis: Honoraria. Hjorth-Hansen:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Porkka:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Mustjoki:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

Dominant genetic aberrations and coexistent EBV infection in HIV-related oral plasmablastic lymphomas

We present common cytogenetic features in the largest cohort of plasmablastic lymphoma (PBL) of the oral cavity published to date. This cohort included 45 patients, 32 of whom had a known HIV status, of which 31 were HIV positive. Ninety eight per cent of all PBL cases were known to be EBV positive. In line with previous studies, we found that rearrangements of the MYC gene was the most common genetic abnormality seen in 60% of cases with the immunoglobulin heavy chain (IGH) locus as a partner in 51% of cases. Additional complex genetic aberrations were frequent, in particular, an increased copy number of the CCND1 gene was seen in 41% of cases with true amplification of CCND1 in 15% of cases. Aneuploidy was also observed for the BCL6 gene in 28% of cases. Interestingly, rearrangements of both IGH genes were detected in 16% of cases with t(14;18) and t(11;14) respectively involved in conjunction with a t(8;14) in two cases. These bi-allelic IGH rearrangements have not been described before in oral PBL. Our results reinforce the notion that EBV infection and MYC rearrangements are important events in the pathogenesis of oral PBL. The genetic diversity and complexity observed in these cases, underlines the importance to genetically characterise PBL patients at presentation as this may inform the choice of more effective treatment modalities.

FancJ/Brip1 helicase protects against genomic losses and gains in vertebrate cells

Defects in the FANCJ/BRIP1 helicase gene are associated with genome instability disorders such as familial breast cancer or Fanconi anemia (FA). Although FANCJ has an in vitro activity to resolve G-quadruplex (G4) structures, and FANCJ ortholog in C. elegans prevents G4-associated deletions during replication, how FANCJ loss affects genome integrity in higher organisms remains unclear. Here, we report that FANCJ, but not other FA genes FANCD2 or FANCC, protected against large-scale genomic deletion that occurred frequently at the rearranged immunoglobulin heavy chain (IgH) locus in chicken DT40 cell line, suggesting that FancJ protects the genome independently of the FA ubiquitination pathway. In a more unbiased approach using array-comparative genomic hybridization, we identified de novo deletions as well as amplifications in fancj cells kept in culture for 2 months. A cluster of G4 sequence motifs was found near the breakpoint of one amplified region, but G4 sequence motifs were not detected at the breakpoints of two deleted regions. These results collectively suggest that, unlike in C. elegans, actions of vertebrate FANCJ to promote genome stability may not be limited to protection against the G4-mediated gene deletions.

CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells

Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V(H) genes to be brought in proximity with D(H)-J(H) segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ~60 sites throughout the V(H) region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5' of DFL16 and the 3' regulatory region, and also the intronic enhancer (Eμ), creating a D(H)-J(H)-Eμ-C(H) domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D(H) region and parts of the V(H) locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.

ATMIN Is Required for Maintenance of Genomic Stability and Suppression of B Cell Lymphoma

SummaryDefective V(D)J rearrangement of immunoglobulin heavy or light chain (IgH or IgL) or class switch recombination (CSR) can initiate chromosomal translocations. The DNA-damage kinase ATM is required for the suppression of chromosomal translocations but ATM regulation is incompletely understood. Here, we show that mice lacking the ATM cofactor ATMIN in B cells (ATMINΔB/ΔB) have impaired ATM signaling and develop B cell lymphomas. Notably, ATMINΔB/ΔB cells exhibited defective peripheral V(D)J rearrangement and CSR, resulting in translocations involving the Igh and Igl loci, indicating that ATMIN is required for efficient repair of DNA breaks generated during somatic recombination. Thus, our results identify a role for ATMIN in regulating the maintenance of genomic stability and tumor suppression in B cells.

ATMIN: A New Tumor Suppressor in Developing B Cells

In the current issue of Cancer Cell , Loizou et al. report that the deletion of ATM Interactor (ATMIN) in developing B cells leads to profound genomic instability and clonal B cell leukemia, revealing a novel tumor suppressor function for ATMIN.

Composite T lymphoblastic leukemia/lymphoma and diffuse large B-cell lymphoma: Case report

This report concerns a unique case of a composite lymphoma composed of T-lymphoblastic leukemia/lymphoma (T-LBL) and diffuse large B-cell lymphoma (DLBCL) in a 72-year-old woman with generalized lymphadenopathy, splenomegaly and ascites. Laboratory findings showed increased lactate dehydrogenase and soluble interleukin-2 receptor. The biopsy specimen showed replacement of the normal architecture of the lymph nodes by a tumor containing a dual cell population composed of large lymphocytes and medium-sized lymphocytes. Sheets of large lymphocytes often were punctuated by clusters of medium-sized lymphocytes. Flow cytometry and immunohistochemical analysis showed a composite lymphoma with both T-LBL and DLBCL. The T-LBL expressed CD1a, CD3, CD4, CD8, and terminal deoxynucleotidyl transferase. The DLBCL expressed CD19 and CD20, CD23, bcl-2, bcl-6, MUM1 and immunoglobulin κ light chain. Polymerase chain reaction detected a monoclonal pattern of T-cell receptor γ and immunoglobulin heavy chain rearrangements in the same specimen. She received eight cycles of R-CHOP (rituximab+cyclophosphamide, doxorubicin, vincristine, prednisone) therapy and achieved complete remission. She has shown no signs of recurrence 20 months after the diagnosis. We describe here a very unusual and, to the best of our knowledge, an as yet never reported case of a primary composite lymphoma of T-LBL and DLBCL.

Increase in Minimal Residual Disease in Peripheral Blood before Clinical Relapse in Dogs with Lymphoma that Achieved Complete Remission after Chemotherapy

We developed previously a minimal residual disease (MRD) monitoring system in dogs with lymphoma by exploring a highly sensitive real-time PCR system. To identify the change in MRD before clinical relapse in dogs with lymphoma that achieved complete remission after chemotherapy. Twenty dogs with multicentric high-grade B-cell lymphoma. MRD levels in peripheral blood mononuclear cells (PBMCs) were measured by real-time PCR amplifying the rearranged immunoglobulin heavy chain gene. MRD measurement and clinical assessment were performed every 2-4 weeks for 28-601 days after completion of chemotherapy. An increase in MRD was defined as an increase by more than 0.5, calculated by log₁₀ [copy number of MRD per 10⁵ PBMCs], based on the uncertainty level observed in a canine lymphoma cell line. During the follow-up period, 15 dogs relapsed in 28-320 days (median, 120 days) after completion of chemotherapy. An increase in MRD was detected 2 weeks or more before relapse in 14 of the 15 dogs, but an increase in MRD before relapse could not be detected in the remaining 1 dog. The time from increased MRD to clinical relapse was 0-63 days (median, 42 days). In contrast, no increase in MRD was detected in 5 dogs that did not experience clinical relapse. An increase in MRD can be detected before clinical relapse in dogs with lymphoma. Application of early reinduction therapy based on an increase in MRD before clinical relapse may improve treatment outcome in canine lymphoma.

Extraoral plasmablastic lymphoma with intravascular component and MYC translocation

Plasmablastic lymphoma (PBL) is an uncommon, clinically aggressive, Epstein-Barr virus-driven B-cell lymphoma that was initially described in tumors of relatively young human immunodeficiency virus-positive men. Subsequent to initial reports, the clinical and pathological spectrum of this disease has been expanded such that, now, PBL is recognized to be a heterogeneous disease entity. Plasmablastic lymphoma has been seen in clinical settings outside those initially reported and has been shown to demonstrate a variety of morphologic patterns. We describe a case of extraoral PBL in an human immunodeficiency virus-infected patient with a computed tomography-identified heterogeneously enhancing mass in the stomach. Histologically, a prominent intravascular component was identified. Fluorescent in situ hybridization analysis for MYC/IGH (immunoglobulin heavy chain) rearrangement t(8;14) identified fusion signals, confirming the presence of MYC rearrangement. The presence of a prominent intravascular in our case is unique. To our knowledge, these findings have not been observed in the previous reports of PBL. The observation of this vascular component supports the heterogeneity of PBL and may be an indicator of tumor aggressiveness. We were able to demonstrate the MYC/IGH rearrangement in our case of PBL. The interplay between Epstein-Barr virus and this MYC rearrangement may be similar to what is observed in Burkitt lymphoma, another clinically aggressive non-Hodgkin lymphoma.

Molecular Remission After Bortezomib-Thalidomide-Dexamethasone Compared with Thalidomide-Dexamethasone as Consolidation Therapy Following Double Autologous Transplantation for Multiple Myeloma: Results of a Qualitative and Quantitative Analysis

AbstractAbstract 861The high rate of complete response (CR) effected by novel agents as induction and consolidation/maintenance therapy in multiple myeloma (MM) has renewed interest in the evaluation of minimal residual disease (MRD) after these combined treatment strategies. For this purpose, a useful molecular marker is represented by the immunoglobulin heavy-chain (IgH) rearrangement which can be carefully detected by means of polymerase chain reaction (PCR). We designed a molecular sub-study to the phase 3 GIMEMA trial of bortezomib-thalidomide-dexamethasone (VTD) vs. thalidomide-dexamethasone (TD) incorporated into double autologous stem cell transplantation (ASCT) for newly diagnosed MM. By study design, patients randomized at diagnosis to VTD or TD as induction therapy before ASCT received two 35-day cycles of consolidation therapy with VTD or TD starting 3 months after ASCT(s), independently from prior response. Doses of study drugs were the following: V, 1.3 mg/m2 once weekly; T, 100 mg/d through d 1 to 70; D, 320 mg/cycle. Aim of the molecular study was to compare the activity of VTD consolidation with that of TD by qualitative and quantitative PCR analysis. Patients who were planned to receive consolidation with either VTD or TD and who were in confirmed CR or near CR before the start of consolidation therapy were eligible for entry into the molecular study. A qualitative and quantitative PCR analysis with patient-specific primers was performed on bone marrow samples collected at day 0 and at day +70 after the two preplanned cycles of either VTD or TD. At this time, 84 patients were included in the study; of these, 67 patients were analyzed for MRD detection based on the availability of: 1) a molecular marker (e.g. IgH rearrangement) assessed at diagnosis; 2) both pre- and post-consolidation bone marrow samples. According to randomization at diagnosis, 35 out of 67 patients received VTD consolidation, while TD consolidation was given to 32 patients. Qualitative PCR analysis performed on bone marrow samples collected at day 0 showed that MRD before the start of VTD consolidation therapy was undetectable in 15 out of 35 patients, or 43%; the corresponding value before the start of TD consolidation was 37.5% (or 12 out of 32 patients). In comparison with the pre-consolidation status, analysis of bone marrow samples collected at day +70 revealed an upgrade in PCR-negativity from 37.5% to 52% with TD consolidation and from 43% to 67% with VTD consolidation (p=0.05, according to McNemar's test). By using the Fisher's exact test, the proportion of patients with post-consolidation PCR-negativity was significantly higher with VTD vs. TD consolidation (p=0.05). These data were furtherly extended by a Real-time quantitative PCR analysis, which could be performed in 45 out of the 67 patients initially included in the study (22 in the TD arm and 23 in VTD). In comparison with residual tumor mass at day 0, quantitative PCR analysis of bone marrow samples collected at day +70 revealed a median 1 log reduction in tumor burden with TD consolidation vs. a median 5 log reduction with VTD consolidation. By using the Wilcoxon test, the overall reduction in residual tumor burden effected by VTD consolidation therapy was statistically significant (p=0.05). In conclusion, in comparison with TD consolidation, two 35-day cycles of consolidation therapy with VTD following double ASCT significantly increased the rate of molecular remissions and significantly reduced the burden of residual myeloma cells persisting after ASCT. Superior activity of VTD over TD consolidation was retained although prior exposure to the same regimen given as induction therapy in preparation for ASCT. Additional data on molecular follow-up analyses of bone marrow samples collected after day +70 and comparisons of the prognostic relevance of PCR-negativity with PCR-positivity, as well as of PCR-negativity induced by VTD with that induced by TD will be presented during the meeting.Supported by: Progetto di Ricerca Finalizzata Orientata (to M.C), BolognAIL, Fondazione del Monte di Bologna e Ravenna. Disclosures:Baccarani:NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Cavo:jansen-cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no; celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no; novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no.

Hematopoietic Stem Cells Are Primarily Involved In Pathogenesis of Chronic Lymphocytic Leukemia.

AbstractAbstract 4604Chronic lymphocytic leukemia (CLL) is characterized by consistent expansion of B cells in peripheral lymphoid organs. CLL B cells express CD5 antigen, and have clonal rearrangement of immunoglobulin heavy chain (IGH) genes with the restricted usage of VH genes. CLL has thus been believed to represent retention or proliferation of abnormal B cell clones presumably with anti-apoptotic potential, or with deregulated response to auto-antigens. In this study, we extensively search for CLL-initiating cells by utilizing the xenogeneic transplantation system, in which human hematopoietic stem cells (HSCs) can normally develop multi-lineage cells including polyclonal B cells. In the xenotransplantation system, neither human (h)CD34-hCD19+ circulating B cells, nor hCD34+hCD38+ bone marrow (BM) progenitor populations from CLL patients engrafted after injection of >106 cells. We then transplanted hCD34+hCD38- BM HSC population from 12 CLL patients. Mice transplanted with as few as 3×103 cells of the hCD34+hCD38- BM population from CLL patients exhibited multi-lineage human hematopoietic reconstitution. Surprisingly, monoclonal or oligoclonal hCD5+ mature CLL-like B cells were reconstituted in the mice transplanted with HSCs from 11 out of 12 CLL patients. IGH gene rearrangement analyses demonstrated that these clonal abnormal B cells derived from hCD34+hCD38- HSCs of CLL patients (CLL-HSCs) were completely independent from the original leukemia. Interestingly, these clonal B cells showed highly biased usage of VH genes for their IGH rearrangement like primary CLL cells. We then checked the chromosomal abnormalities including del13q14 and del11q22 in CLL-HSCs and the clonal B cells reconstituted in the recipient mice. In 3 cases examined, both of them were negative for the chromosomal abnormalities detected in the original CLL cells, suggesting that the chromosomal abnormalities were acquired as a 2nd leukemogeneic events during CLL progression in the patients.The fact that CD34+CD38- HSCs from CLL patients but not those from normal individuals reconstitute monoclonal B cell lymphocytosis (MBL)-like human hematopoiesis in our xenograft model strongly suggests that some genetic abnormalities for mature monoclonal B cell development are acquired already at the HSC level in CLL patients. HSCs in CLL patients are multipotent, but once they commit to the B cell lineage, they use preferentially the VH1, VH3 and VH4 regions for IGH recombination and MBL-like clonal B cell expansion occur. Our hypothesis is that such B cell clones may already be abnormal in that they clonally expand in response, for example, to auto-antigens (xeno-antigens in mice), and these preleukemic B cell clone may possibly sequentially receive additional genetic events including chromosomal abnormalities during development of CLL and finally progress to clinical CLL.Although this xenograft model may not recapitulate full picture of CLL progression, our data clearly show that primary leukemogenic event occurs at the multipotent HSC stage in human CLL. Disclosures:No relevant conflicts of interest to declare.

Molecular characterization of paediatric idiopathic hypereosinophilia

The hypereosinophilic syndromes (HES) include a group of heterogeneous diseases characterized by the persistent increase of the number of eosinophils in blood and bone marrow. Few cases of paediatric hypereosinophilia (pHES) have been described in the literature. Early identification of pHES that may evolve towards a lymphomyeloproliferative disease is relevant in light of prognostic and therapeutic implications. Molecular features of 10 pHES patients were analysed at presentation and during their clinical course, including analysis of BCR-ABL1 and FIP1L1/PDGFRA fusion genes, quantitation of WT1 gene copy number and clonality of T-cell receptor (TCR) and immunoglobulin heavy chain (IGH). All patients had normal karyotype and germline TCR configuration. Five children showed IGH clonality at presentation: of these, two developed a B non-Hodgkin lymphoma and a B-lineage acute lymphocytic leukaemia at six and 12 months respectively, two spontaneously reverted to a polyclonal IGH profile during the follow-up, and the last one persisted with pHES without B-clonal evolution after 19 months. One patient had a PDGFRA/FIP1L1 fusion and achieved hematologic and molecular remission after imatinib therapy. IGH rearrangement was observed to be a frequent molecular feature of pHES and may precede B-cell clonal expansion and evolution into B-cell malignancies in children.

Clinical significance of clonality and Epstein‐Barr virus infection in adult patients with hemophagocytic lymphohistiocytosis

We assessed the clinical significance of T or B cell clonality and Epstein-Barr virus (EBV) infection in adult patients with hemophagocytic lymphohistiocytosis (HLH) to identify factors related to prognosis. A total of 30 adult patients with diagnosed HLH were included in the study. In all patients, EBV-DNA in peripheral blood was examined by quantitative real-time polymerase chain reaction and bone marrow cells were examined for clonal rearrangement of T cell receptor gamma(TCRG) and immunoglobulin heavy chain (IGH) genes. TCRG clones were detected in 10 patients (33.3%) and IGH clones were detected in 8 patients (26.7%). We found no correlation between clonality and patient outcome. The patients less than 1,000 copies (mL)21 of EBVDNA showed a significantly higher clinical response (P 5 0.008) and longer overall survival (P 5 0.01) than those with high viral load of EBV-DNA. Our results suggest that TCRG and IGH rearrangement do not have any clinical significance in adult patients with HLH, but that high viral load of EBV-DNA may be a risk factor for poor outcomes. In HLH, high viral load of EBV-DNA should thus suggest a prompt approach with aggressive therapeutic interventions.

Posttransplantation lymphoproliferative disease involving the pituitary gland

Posttransplantation lymphoproliferative disorders (PTLD) are heterogeneous lesions with variable morphology, immunophenotype, and molecular characteristics. Multiple distinct primary lesions can occur in PTLD, rarely with both B-cell and T-cell characteristics. Lesions can involve both grafted organs and other sites; however, PTLD involving the pituitary gland has not been previously reported. We describe a patient who developed Epstein-Barr virus-negative PTLD 13 years posttransplantation involving the terminal ileum and pituitary, which was simultaneously involved by a pituitary adenoma. Immunohistochemistry of the pituitary lesion showed expression of CD79a, CD3, and CD7 with clonal rearrangements of both T-cell receptor gamma chain (TRG@) and immunoglobulin heavy chain (IGH@) genes. The terminal ileal lesion was immunophenotypically and molecularly distinct. This is the first report of pituitary PTLD and illustrates the potentially complex nature of PTLD.

EBV-positive diffuse large B-cell lymphoma of the elderly with dual genotype and aberrant TIA-1 expression: a case report and review of literature

B-cell lymphomas generally do not express T-cell intracellular antigen-1 (TIA-1), a cytotoxic granule-associated protein. We report a case of EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly with aberrant expression of TIA-1. The patient was an 84-year-old man with a history of progressively enlarged left parotid mass and mild facial paresis for 2 months. The incisional biopsy revealed sheets of medium to large lymphoma cells with focal angiocentricity and extensive tumor necrosis; only a few scattered small lymphocytes were noted among the lymphoma cells. Immunohistochemistry in formalin-fixed paraffin-embedded tissue demonstrated that the lymphoma cells marked with CD20, CD30, CD45 (focal), CD79a, BCL2 (focal and weak), PAX5, Oct2, BOB.1, IRF4/MUM1, EBV-LMP1, and TIA-1, but they did not mark with CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD23, CD56, CD57, CD138, kappa and lambda immunoglobulin light chains, BCL6, βF1, granzyme B, or perforin. The aberrant TIA-1 expression was then confirmed by two different autostainers. EBV-encoded RNA (EBER) in situ hybridization was positive in the lymphoma cells. Gel electrophoresis for immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements revealed no definite clonal band, but fluorescence capillary electrophoresis demonstrated positive results for both IgH and TCR genes. Finally, we concluded this case as an EBV-positive DLBCL of the elderly with dual genotype (rearrangements of both IgH and TCR genes) and aberrant TIA-1 protein expression.

Polymerase chain reaction (PCR) detection of B cell clonality in Sjögren's syndrome patients: a diagnostic tool of clonal expansion

Sjögren's syndrome (SS) is an autoimmune disease characterized by clonal B cell attack of the exocrine glands and dysregulated expression of B cell-activating factor (BAFF). Based upon the current data of increased rates of lymphoid malignancy, as non-Hodgkin's lymphoma (NHL) is associated with SS, we propose the detection of clonal rearrangements of immunoglobulin heavy chain (IgH) gene in those patients as a predictor of malignant clonal expansion. To test our proposal, we examined the IgH clonal rearrangements in SS patients (60) and healthy control subjects (42) having chronic non-specific sialadenitis, to determine the presence of clonal B cells in minor labial salivary glands (MSG) of SS patients. Clonal B cell expansion was assessed by two polymerase chain reaction (PCR) assays: (i) semi-nested PCR, against sequences encoding framework regions FR3, FR2 and FR1c of the variable chain IgH gene in B cells present in the MSG infiltrate; and (ii) the PCR-enzyme-linked immunosorbent assay (ELISA) technique, against the major and minor breakpoint regions of the Bcl-2 oncogene coupled with a variable segment of the IgH to assess the Bcl-2/JH translocation. When FR3, FR2 and FR1c primers were employed, we detected B cell monoclonality in 87% of the SS patients and 19% of the control subjects. The association between inflammation severity of the MSG pattern and the presence of B cell clonality was found to be statistically significant (P<0.01). We concluded that the presence of B cell clonality in MSG can be used as a index of an altered microenvironment favouring the development of lymphoma in SS patients.