Major Breakpoint Region Research Articles

IGH::NSD2 Fusion Gene Transcript as Measurable Residual Disease Marker in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological malignancy. Approximately 15% of MM patients are affected by the t(4;14) translocation resulting in the IGH::NSD2 fusion transcript. Breakage occurs in three major breakpoint regions within the NSD2 gene (MB4-1, MB4-2, and MB4-3), where MB4-1 leads to the production of full-length protein, while truncated proteins are expressed in the other two cases. Measurable residual disease (MRD) has been conclusively established as a crucial prognostic factor in MM. The IGH::NSD2 fusion transcript can serve as a sensitive MRD marker. Using bone marrow (BM) and peripheral blood (PB) samples from 111 patients, we developed a highly sensitive quantitative real-time PCR (qPCR) and digital PCR (dPCR) system capable of detecting fusion mRNAs with a sensitivity of up to 1:100,000. PB samples exhibited sensitivity three orders of magnitude lower compared to BM samples. Patients with an MB4-2 breakpoint demonstrated significantly reduced overall survival (p = 0.003). Our novel method offers a simple and sensitive means for detecting MRD in a substantial proportion of MM patients. Monitoring may be carried out even from PB samples. The literature lacks consensus regarding survival outcomes among patients with different NSD2 breakpoints. Our data align with previous findings indicating that patients with the MB4-2 breakpoint type tend to exhibit unfavorable overall survival.

Correlation of t(14;18) translocation breakpoint site with clinical characteristics in follicular lymphoma.

t(14;18)(q32;q21) translocation is an important genetic feature of follicular lymphoma resulting in antiapoptotic B-cell lymphoma 2 (BCL2) protein overexpression. On chromosome 18 breakpoint-site variation is high but does not affect BCL2. Breakpoint most commonly occurs at major breakpoint region (MBR) but may happen at minor cluster region (mcr) and between MBR and mcr at 3'MBR and 5'mcr. The aim of this study was to analyze the correlation of t(14;18)(q32;q21) breakpoint site with clinical characteristics in follicular lymphoma. We included patients diagnosed with follicular lymphoma who received at least 1 cycle of systemic treatment and had the t(14;18)(q32;q21) translocation detected by polymerase chain reaction (PCR) at MBR, mcr or 3'MBR prior to first treatment. Among patients with different breakpoints, sex, age, disease grade, stage, B-symptoms, follicular lymphoma international prognostic index (FLIPI), presence of bulky disease, progression free survival and overall survival were compared. Of 84 patients, 63 had breakpoint at MBR, 17 at mcr and 4 at 3'MBR. At diagnosis, the MBR group had a significantly lower disease stage than the mcr group. Although not significant, in the MBR group we found a higher progression-free survival (PFS) and overall survival (OS), lower grade, age, FLIPI, and less B-symptoms. Compared to patients with mcr breakpoint, those with MBR breakpoint seem to be characterised by more favourable clinical characteristics. However, a larger study would be required to support our observation.

A putative G-quadruplex structure in the proximal promoter of VEGFR-2 has implications for drug design to inhibit tumor angiogenesis

Tumor angiogenesis is mainly regulated by vascular endothelial growth factor (VEGF) produced by cancer cells. It is active on the endothelium via VEGF receptor 2 (VEGFR-2). G-quadruplexes are DNA secondary structures formed by guanine-rich sequences, for example, within gene promoters where they may contribute to transcriptional activity. The proximal promoter of VEGFR-2 contains a G-quadruplex, which has been suggested to interact with small molecules that inhibit VEGFR-2 expression and thereby tumor angiogenesis. However, its structure is not known. Here, we determined its NMR solution structure, which is composed of three stacked G-tetrads containing three syn guanines. The first guanine (G1) is positioned within the central G-tetrad. We also observed that a noncanonical, V-shaped loop spans three G-tetrad planes, including no bridging nucleotides. A long and diagonal loop, which includes six nucleotides, connects reversal double chains. With a melting temperature of 54.51 °C, the scaffold of this quadruplex is stabilized by one G-tetrad plane stacking with one nonstandard bp, G3-C8, whose bases interact with each other through only one hydrogen bond. In summary, the NMR solution structure of the G-quadruplex in the proximal promoter region of the VEGFR-2 gene reported here has uncovered its key features as a potential anticancer drug target.

DNA structural basis for fragility at peak III of BCL2 major breakpoint region associated with t(14;18) translocation

Maintaining genome integrity is crucial for normal cellular functions. DNA double-strand breaks (DSBs), when unrepaired, can potentiate chromosomal translocations. t(14;18) translocation involving BCL2 gene on chromosome 18 and IgH loci at chromosome 14, could lead to follicular lymphoma. Molecular basis for fragility of translocation breakpoint regions is an active area of investigation. Previously, formation of non-B DNA structures like G-quadruplex, triplex, B/A transition were investigated at peak I of BCL2 major breakpoint region (MBR); however, it is less understood at peak III. In vitro gel shift assays show faster mobility for MBR peak III sequences, unlike controls. CD studies of peak III sequences reveal a spectral pattern different from B-DNA. Although complementary C-rich stretches exhibit single-strandedness, corresponding guanine-rich sequences do not show DMS protection, ruling out G-quadruplex and triplex DNA. Extrachromosomal assay indicates that peak III halts transcription, unlike its mutated version. Taken together, multiple lines of evidence suggest formation of potential cruciform DNA structure at MBR peak III, which was also supported by in silico studies. Thus, our study reveals formation of non-B DNA structure which could be a basis for fragility at BCL2 breakpoint regions, eventually leading to chromosomal translocations.

Occupational exposure to pesticides and occurrence of the chromosomal translocation t(14;18) among farmers in Jordan.

An increased incidence of non-Hodgkin's lymphoma (NHL) has been reported in farmers and other occupational groups working with pesticides. In these individuals, an increased prevalence of the chromosomal translocation t(14;18)(q32;q21), one of the most common chromosomal abnormalities in NHL, has been detected in peripheral blood lymphocytes. This translocation juxtaposes the antiapoptotic BCL2 protein to the immunoglobulin heavy chain gene locus (IGH) leading to overexpression of BCL2. This causes an increase in cell survival, paving the way for malignant transformation. The present study aimed to evaluate the association between the occurrence of the chromosomal translocation t(14;18) and occupational exposure to pesticides among a group of Jordanian farmers. A total of 192 male subjects including 96 agricultural workers and 96 control subjects participated in this study. BCL2-IGH t(14;18) fusions were detected by a nested polymerase chain reaction (PCR) assay targeting the major breakpoint region (MBR). We found that occupational exposure to pesticides in open-field farming and insecticide used on animals increased the frequency of the chromosomal translocation t(14;18). Farmers occupationally exposed to pesticides and insecticide were 13.5 times more likely to harbor t(14;18). 63.5% (61 of 96) of farmers compared to 11.5% (11 of 96) of controls carried the translocation (odds ratio: 13.5; 95% confidence interval (CI)=6.3-28.6). We ruled out the influence of possible confounding factors such as age, duration of sun exposure, alcohol intake, smoking, and use of personal protective equipment. Our results indicate that pesticides increased the frequency of chromosomal translocation in the 14q32 region. Accordingly, the presented data agrees with previous suggestions from the literature that pesticides might be involved in the development of NHL through the t(14;18) pathway.

Initial diagnosis of chronic myelogenous leukemia based on quantification of M-BCR status using droplet digital PCR.

Formed from a reciprocal translocation t(9:22)(q34;q11) of genetic material between the long arms of human chromosomes 9 and 22, the constitutively active breakpoint cluster region (BCR) Abelson 1 (ABL) tyrosine kinase BCR-ABL is known to be causative of chronic myelogenous leukemia (CML). In 98% of CML patients harboring the t(9:22)(q34;q11) translocation, known as the Philadelphia chromosome, the chimeric BCR-ABL oncogene is created through cleavage of the BCR gene within its major breakpoint region (M-BCR) and breakage of the ABL gene within a 100-kbp region downstream of exon 2a. Clinical detection of the fused BCR-ABL oncogene currently relies on direct visualization by fluorescence in situ hybridization (FISH), a relatively tedious assay that typically offers a detection limit of ca. 2%. Here, we describe a novel assay that uses droplet digital PCR (ddPCR) technology to reliably measure M-BCR status and the presence of BCR-ABL. When applied to cell-line models of CML, the assay accurately quantifies BCR-ABL frequency to a detection limit of 0.25%. It therefore offers improved specificity relative to FISH, and may allow identification of variant translocation patterns, including derivative chromosome 9 deletions.

Determinants of the t(14;18) translocation and their role in t(14;18)-positive follicular lymphoma.

The strong association between t(14;18) translocation and follicular lymphoma (FL) is well known. However, the determinants of this chromosomal aberration and their role in t(14;18) associated FL remain to be established. t(14;18) frequency within the B cell lymphoma 2 major breakpoint region was determined for 135 incident FL cases and 251 healthy controls as part of a nested case-control study within the European Prospective Investigation into Cancer cohort. Quantitative real-time PCR was performed in DNA extracted from blood samples taken at recruitment. The relationship between prevalence and frequency of the translocation with baseline anthropometric, lifestyle, and dietary factors in cases and controls was determined. Unconditional logistic regression was used to explore whether the risk of FL associated with these factors differed in t(14;18)(+) as compared to t(14;18)(-) cases. Among incident FL cases, educational level (χ(2) p = 0.021) and height (χ(2) p = 0.025) were positively associated with t(14;18) prevalence, and cases with high frequencies [t(14;18)(HF)] were significantly taller (t test p value = 0.006). These findings were not replicated in the control population, although there were a number of significant associations with dietary variables. Further analyses revealed that height was a significant risk factor for t(14;18)(+) FL [OR 6.31 (95% CI 2.11, 18.9) in the tallest versus the shortest quartile], but not t(14;18)(-) cases. These findings suggest a potential role for lifestyle factors in the prevalence and frequency of the t(14;18) translocation. The observation that the etiology of FL may differ by t(14;18) status, particularly with regard to height, supports the subdivision of FL by translocation status.

Methylation of CpG sites in BCL2 major breakpoint region and the increase of BCL2/JH translocation with aging.

The BCL2 breakage mechanism has been shown to be highly dependent on DNA methylation at the major breakpoint region (MBR) CpG sites. We recently described an increased frequency of BCL2/ JH translocation with aging. It is known that methylation levels change with aging. The present study aimed to determine whether methylation alterations at CpG sites of BCL2 MBR were the cause of increased breakages with aging. We analyzed the methylation levels of three CpG sites on the region by pyrosequencing and studied if methylation levels and/or polymorphisms affecting CpG sites were associated with an increase of translocations. We observed that although the methylation levels of MBR CpG sites were higher in individuals with BCL2/JH translocation, in contrast to our expectations, these levels decreased with the age. Moreover, we show that polymorphisms at those CpG sites leading to absence of methylation seem to be a protective factor for the apparition of translocations.

Minimal Residual Disease Detection by Droplet Digital PCR in Multiple Myeloma, Mantle Cell Lymphoma, and Follicular Lymphoma: A Comparison with Real-Time PCR

Real-time quantitative PCR (qPCR) is a well-established tool for minimal residual disease (MRD) detection in mature lymphoid malignancies. Despite remarkable sensitivity and specificity, qPCR has some limitations, particularly in the need for a reference standard curve, based on target serial dilutions. In this study, we established droplet digital PCR (ddPCR) for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma and compared it head-to-head with qPCR. We observed that ddPCR has sensitivity, accuracy, and reproducibility comparable with qPCR. We then compared the two approaches in 69 patients with a documented molecular marker at diagnosis (18 multiple myelomas, 21 mantle cell lymphomas assessed with the immunoglobulin gene rearrangement, and 30 follicular lymphomas with the use of the BCL2/immunoglobulin gene major breakpoint region rearrangement). ddPCR was successful in 100% of cases, whereas qPCR failed to provide a reliable standard curve in three patients. Overall, 222 of 225 samples were evaluable by both methods. The comparison highlighted a good concordance (r=0.94, P<0.0001) with 189 of 222 samples (85.1%; 95% CI, 80.4%-89.8%) being fully concordant. We found that ddPCR is a reliable tool for MRD detection with greater applicability and reduced labor intensiveness than qPCR. It will be necessary to authorize ddPCR as an outcome predictor tool in controlled clinical settings and multilaboratory standardization programs.

Detection of G-quadruplex DNA using primer extension as a tool.

DNA sequence and structure play a key role in imparting fragility to different regions of the genome. Recent studies have shown that non-B DNA structures play a key role in causing genomic instability, apart from their physiological roles at telomeres and promoters. Structures such as G-quadruplexes, cruciforms, and triplexes have been implicated in making DNA susceptible to breakage, resulting in genomic rearrangements. Hence, techniques that aid in the easy identification of such non-B DNA motifs will prove to be very useful in determining factors responsible for genomic instability. In this study, we provide evidence for the use of primer extension as a sensitive and specific tool to detect such altered DNA structures. We have used the G-quadruplex motif, recently characterized at the BCL2 major breakpoint region as a proof of principle to demonstrate the advantages of the technique. Our results show that pause sites corresponding to the non-B DNA are specific, since they are absent when the G-quadruplex motif is mutated and their positions change in tandem with that of the primers. The efficiency of primer extension pause sites varied according to the concentration of monovalant cations tested, which support G-quadruplex formation. Overall, our results demonstrate that primer extension is a strong in vitro tool to detect non-B DNA structures such as G-quadruplex on a plasmid DNA, which can be further adapted to identify non-B DNA structures, even at the genomic level.

A Comparative Analysis Of Next-Generation Sequencing and Real-Time Quantitative PCR For Minimal Residual Disease Detection In Follicular Lymphomas

BackgroundDetection of minimal residual disease (MRD) by t(14;18) Real-Time Quantitative (RQ) PCR is an important tool during clinical follow-up in patients (pts) with follicular lymphoma (FL). However, only the major breakpoint region (MBR) and minor cluster region (mcr) breakpoints have been currently exploited as MRD targets. Therefore, only 50% to 65% of pts can be assessed by t(14;18) RQ-PCR. Alternative targets such as the immunoglobulin heavy chain variable region (IGH) can be used only with limitations due to somatic hypermutation. IGH-based next-generation sequencing (NGS) might provide an alternative approach and provide increased sensitivity, specificity, accuracy and reproducibility. We performed a comparison of the two approaches in 29 FL pts. Patients and MethodsOverall, 206 samples (85 bone marrow, 114 peripheral blood, 6 stem cell aliquots and one lymph node sample) were investigated from 29 FL pts in which RQ-PCR based MRD analysis had been performed in the context of prospective clinical trials. Overall, 33 dx and 173 follow-up (FU) samples were analyzed. 23/29 pts had a PCR detectable t(14;18) rearrangement, 5 pts had a clonal IGH rearrangement only and one patient had no marker by consensus PCR. NGS was performed at Sequenta in South San Francisco. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from genomic DNA. Amplified products were sequenced to obtain a high degree of coverage and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency in the dx sample and then quantitated in FU samples.Overall comparability of MRD results by RQ-PCR and NGS was assessed using correlation analysis. A positive/negative discordance between two results was defined as major when the positive result was >1E-05 and minor when ≤1E-05; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. ResultsAll 29 pts were evaluable with at least one method. IGH NGS identified an index clone in 22/29 cases. Seven patients pts were only quantifiable by t(14;18) RQ-PCR while IGH NGS did not identify an index clone for sequencing. In all but one dx sample demonstrated low level lymphoma infiltration with MRD level below 10-3. In one of these cases, IG kappa could be successfully sequenced for MRD indicating that not only low level MRD but also somatic mutation of IGH is a potential pitfall for MRD detection by NGS in FL.15 pts were evaluable for MRD by t(14;18) RQ-PCR and NGS. In 97 FU samples, a significant concordance between MRD methods could be demonstrated (r2=0.80) (p<0.0001). Of these samples, 44 were MRD positive and 45 were MRD negative with both tools. A quantitative discordance occurred in 12/44 MRD positive samples, where MRD was higher in 7 samples and lower in 5 samples by NGS. A major discordance occurred in 4 samples where t(14;18) RQ-PCR was positive and NGS was negative. A minor discordance was detectable in 4 samples where in 2 samples t(14;18) RQ-PCR was positive and NGS was negative, and in the other 2 samples the opposite was correct.In 5 t(14;18) negative cases and one with an unusual large t(14;18) rearrangement MRD was quantified by IGH-RQ-PCR using cloned plasmids and IGH NGS. In all cases both methods also showed excellent concordance.One advantage of the IGH NGS approach is its ability to detect different IGH sequences generated by the somatic hypermutation process. We have seen in some patients a dynamic picture of the IGH clonotypes with the rise and fall of different clonotypes at different time points (Fig 1). [Display omitted] ConclusionsNGS represents a feasible tool for IGH-based MRD monitoring that allows analysis of a larger group of FL pts. Our results show that the two methods have a high level of correlation. Lymphoma infiltration of dx samples and somatic mutation of IGH is a critical point for identification of the tumor-specific clonotypes by NGS, therefore different MRD methods should complement each other to allow MRD assessment for the majority of pts. Furthermore IGH NGS sequencing has the potential to detect and track IGH evolution in FL. Disclosures:Kneba:Roche: Consultancy, Research Funding. Faham:Sequenta, Inc.: Employment, Patents & Royalties.

Treatment Of Early Stage Follicular Lymphoma With Involved Field Radiotherapy and Rituximab. Role Of Bcl-2 Molecular Monitoring

BackgroundStage I or II follicular lymphoma (FL) is an uncommon disease, representing only 20% of FL. Conventional treatment is represented by local radiotherapy (RT), which allows eradication of the disease in about 50% of patients. Despite the negative bone marrow biopsy in all cases, most patients present Bcl-2 rearranged cells in the bone marrow (BM) and/or peripheral blood (PB). The aim of this study was to analyze the prognostic role of Bcl-2 molecular monitoring in a series of stage I-II FL cases followed at a single center. MethodsFifty-seven consecutive patients with a confirmed diagnosis of stage I/II FL were investigated at presentation by PCR in order to identify the presence of Bcl-2 rearranged cells in the BM and/or PB. All patients were treated with involved field RT (30-36 Gy). Subsequently, minimal residual disease (MRD) was evaluated every 6 months after RT in patients positive at baseline; patients negative at baseline were not retested. In part of the patients (after 2005) Rituximab was administered in case of persistently positive Bcl-2 cells in the BM or PB after radiotherapy. The PCR analysis of the Bcl-2/IgH rearrangement was performed according to published methods. It consists in a nested PCR that uses in the first round a couple of primers for the major breakpoint region (MBR) or for the minor cluster region (mcr). After this first step, the amplification products were re-amplified using oligonucleotide primers internal to the original ones. An aliquot of the PCR products was analysed on 2% agarose gel containing ethidium bromide in Tris-borate electrophoresis buffer and visualized under UV light. For MBR and mcr, a reproducible sensitivity level of 10-5 and 10-4 respectively, was obtained. Results1. Prognostic value of basal PCR in BM/PB: PCR analysis revealed Bcl-2 rearranged cells in the PB and/or BM in 38/57 patients (66.7%) at presentation. After a median follow-up of 55 months, 11 patients (19.3%) had a clinical relapse; of them, 10 belonged to the group with positive PCR at baseline, while only 1 patient with negative basal Bcl-2 (1.7%) experienced a clinical relapse (Pearson's chi2= 0.058, Fisher exact test = 0.079). Among the 11 patients who showed a clinical relapse, 5 presented a positive Bcl-2 at relapse, 3 were negative (1 already at baseline),while in 3 this information is not evaluable. 2. Effect of local RT: After irradiation of the sole site of the disease, Bcl-2 rearranged cells disappeared in 19 of 38 patients positive at baseline (50%). In 17/38 (44.7%), MRD remained positive, while 2 patients refused to perform the analysis. A negative MRD after RT does not seem to correlate with a lower relapse probability. Only 1 patient died of breast cancer. 3. Effect of rituximab treatment in Bcl-2+ patients: Fourteen patients with persistently positive Bcl-2 after RT were treated with Rituximab 375 mg/m2 for 4 weekly administrations: 9 of them (64%) patients became negative. This result was only temporary in 4/9 cases (1 clinical relapse). Among persistently Bcl-2 positive patients after Rituximab, 1 clinical relapse was also observed. ConclusionsIn limited stage FL, despite a negative BM biopsy, Bcl-2/IgH rearranged cells can be found in the BM and/or PB, and they can disappear after local RT of the involved lymph node(s) in 50% of cases (19/38). The basal presence of Bcl-2+ cells in the BM/PB has a prognostic role: no clinical relapses were observed in Bcl-2 negative cases at baseline, except for 1 patient. Conversely, a negative MRD after radiotherapy does not seem to correlate with a better prognosis. -Rituximab therapy can induce a negativization of Bcl-2 in MRD-positive patients. Nevertheless, Rituximab treatment was only partially effective: negativization was observed in the majority of MRD-positive patients, but it was only temporary in a proportion of them. In Rituximab-treated patients, clinical relapses occurred only in the presence of MRD. -Not all clinical relapses were preceded by MRD positivity; further data are necessary to establish the usefullness of MRD monitoring over time. Prognosis of patients with early-stage FL treated with local RT + Rituximab in case of MRD persistence, is excellent: cause-specific survival=100%, EFS=70% projected at 10 years. Disclosures:No relevant conflicts of interest to declare.

MLL genomic DNA Breakpoints In Infant Acute Leukemia

BackgroundInfant acute leukemia is characterized by high incidence of MLL gene rearrangements. PurposeTo evaluate the distribution of MLL genomic DNA breakpoints and their relation to several diagnostic parameters among infant acute leukemia. Methods72 infants with MLL-rearranged acute lymphoblastic leukemia (ALL) (n=52), acute myeloid leukemia (AML) (n=19) and mixed phenotype acute leukemia (n=1) were included in this study based on the availability of DNA material at diagnosis. In the observed group there were 28 boys (39%) and 44 girls (61%) with median age of 4.9 mo (range 0.03-11.9). Genomic DNA breakpoint detection in MLL gene and translocation partner genes (TPG) was performed by long-distance inverse PCR (LDI-PCR). Exon-intron numbering of MLL gene was done according to I. Nilson et al, 1996. ResultsMajority of ALL cases (n=28; 54%) was characterized by presence of MLL-AF4 fusion gene (FG), less frequently MLL-MLLT1 (n=12; 23%), MLL-MLLT3 (n=7; 13%) and others were found (Table 1). The most common breakpoint location within MLL gene in ALL patients was intron 11, detected in 25 cases (48%). The highest variability of MLL breakpoints was found in MLL-AF4-positive patients: only 11 of 28 (39%) had breakpoints in intron 11. The most stable pattern of MLL genomic DNA breakpoints was observed in MLL-MLLT1-positive patients: 8 of 12 (67%) had breakpoints in intron 11. In AML patients two the most prevalent FGs were MLL-MLLT3 (n=7, 37%) and MLL-MLLT10 (n=5, 26%). The remaining ones are listed in Table 1. The most frequent breakpoints location was intron 8 (8 out of 19, 42%). The most stable pattern was revealed for MLL-MLLT10 FG: MLL breakpoints in 4 of 5 (80%) cases were found in intron 9 (Table 1). ALL patients who had breakpoints in intron 11 were significantly younger (median 3.0 mo, range 0.03-11.6) than all others (median 5.6 mo, range 0.7-11.9) (p=0.025) and than patients with MLL breakpoints in intron 9 (median 6.6 mo, range 3.1-11.9) (p=0.017). For AML cases we did not find any relation between age and breakpoints locations. Distribution of MLL DNA breakpoints was similar in boys and girls and did not depend on type of TPG. Genetic recombinations involving MLL gene predominantly resulted in reciprocal chromosomal translocations (n=62; 86%). Beside them, 6 (11%) insertions were identified in all MLL-MLLT10-positive cases and MLL-SEPT6-positive one. In 11 (15%) patients we found breakpoints within the regions located from 0.7 Kb to 25.4 Kb 3' of the first exon of TPGs (MLLT1 n=9; EPS15 n=1; MYO1F n=1), however fusion transcripts at cDNA level were identified and sequenced in all these cases, indicating a spliced fusion mechanism. 3-way translocations were found in 5 patients and in 1 case we found combination of insertion with interstitial deletion of chromosome 11. The list of reciprocal genes involved in these 6 cases was as follows: CEP164, DNAH6, DCPA1, MCL1 as well as non-coding regions of 2q21.2 and 2p21. We also analyzed breakpoints in TPGs. Except above mentioned spliced fusion cases, the remaining 3 breakpoints in MLLT1 as well as 3 of 4 breakpoints in EPS15 and all breakpoints in MLLT11 were within intron 1 of corresponding genes. In AF4 the major breakpoint region included intron 3 (n=19), intron 4 (n=6) and intron 5 (n=2). We also revealed 2 rare breakpoints in intron 6 and 10. In MLLT3 the most frequent breakpoint location was intron 5 (n=12), additionally 2 cases in intron 5 were identified. In MLLT10 two separate breakpoint locations were found: intron 3 (n=1) and intron 8 (n=3) in combination with intron 9 (n=1). We estimated prognostic significance of MLL breakpoint locations in 31 cases of infant ALL treated by MLL-Baby protocol. 3-year cumulative incidence of relapse was remarkably higher in patients with breakpoints in intron 11 (n=18) in comparison to patients with breakpoint localized from intron 7 to exon 11, inclusively (n=13) (0.85±0.01 and 0.57±0.02, respectively), although difference between these two groups did not achieve statistical significance (p=0.261). Median follow-up time in the observed group was 30 months (range 6–42). ConclusionIn the current study we estimated clinical and prognostic significance of MLL and TPG genomic DNA breakpoints in infant acute leukemia. Our data provide additional information of molecular genetic features of MLL-rearranged infant acute leukemia. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Both CpG Methylation and Activation-Induced Deaminase Are Required for the Fragility of the Human bcl-2 Major Breakpoint Region: Implications for the Timing of the Breaks in the t(14;18) Translocation

The t(14;18) chromosomal translocation typically involves breakage at the bcl-2 major breakpoint region (MBR) to cause human follicular lymphoma. A theory to explain the striking propensity of the MBR breaks at three CpG clusters within the 175-bp MBR region invoked activation-induced deaminase (AID). In a test of that theory, we used here minichromosomal substrates in human pre-B cell lines. Consistent with the essential elements of the theory, we found that the MBR breakage process is indeed highly dependent on DNA methylation at the CpG sites and highly dependent on the AID enzyme to create lesions at peak locations within the MBR. Interestingly, breakage of the phosphodiester bonds at the AID-initiated MBR lesions is RAG dependent, but, unexpectedly, most are also dependent on Artemis. We found that Artemis is capable of nicking small heteroduplex structures and is even able to nick single-base mismatches. This raises the possibility that activated Artemis, derived from the unjoined D to J(H) DNA ends at the IgH locus on chromosome 14, nicks AID-generated TG mismatches at methyl CpG sites, and this would explain why the breaks at the chromosome 18 MBR occur within the same time window as those on chromosome 14.

Expression of Myc, but not pSTAT3, is an adverse prognostic factor for diffuse large B-cell lymphoma treated with epratuzumab/R-CHOP

AbstractSTAT3 regulates cell growth by up-regulating downstream targets, such as Myc. The frequency of phosphorylated STAT3 (pSTAT3) and Myc expression and their prognostic relevance is unknown within diffuse large B-cell lymphoma (DLBCL) germinal center B-cell (GCB) and non-GCB subtypes. pSTAT3 and Myc were studied by immunohistochemistry (IHC) on tumors from 40 DLBCL patients uniformly treated on a clinical trial of epratuzumab/rituximab-CHOP. A total of 35% of cases were pSTAT3-positive, and pSTAT3 positivity was more frequent in the non-GCB (P = .06) type but did not correlate with event-free survival (EFS). Myc expression was observed in 50% of cases and was more frequent in non-GCB type (P = .07). Myc-positive cases had inferior EFS in all patients, including the GCB and pSTAT3-positive cases, were more likely to express Myc (P = .06). Myc translocations involving the major breakpoint regions were found in 10% (3 of 29) of cases, and all 3 cases were GCB and had an inferior EFS (P = .09). pSTAT3, but not Myc expression, was correlated with elevated pretreatment serum cytokines, such as IL-10 (P = .05), G-CSF (P = .03), and TNF-α (P = .04). pSTAT3 IHC in DLBCL tumors has the potential to identify patients for STAT3 pathway–directed therapy; Myc IHC is a potential marker for inferior EFS in GCB patients.

Expression of MYC but Not pSTAT3 Is an Adverse Prognostic Factor for Diffuse Large B Cell Lymphoma (DLBCL) Treated with Epratuzumab/R-CHOP

AbstractAbstract 1575We recently demonstrated in a phase II study that the anti-CD22 monoclonal antibody epratuzumab added to RCHOP improved DLBCL survival parameters. Despite these advances, 30–40% of DLBCL patients still relapse and die of disease. Recent advances have led to the identification of a variety of intracellular oncogenic pathways as potential targets for lymphoma therapy. Specifically, many studies have found that activation of the Signal transducer and activator of transcription 3 (STAT3) pathway promotes tumor cell survival in various types of cancers. STAT3 is a transcription factor and exerts its anti-apoptototic effect through several downstream targets such as MYC. MYC protein can be expressed in lymphoma cells with or without the presence of MYC translocation. The frequency of phosphorylated STAT3 (pSTAT3) and MYC expression and their prognostic relevance are unknown within diffuse large B-cell lymphoma (DLBCL), germinal center B-cell (GCB) and non-GCB subtypes. This study studied the tumor cell expression of pSTAT3 and MYC by IHC paired with serum cytokine levels in a DLBCL patient population uniformly treated on N0489.DLBCL tumor samples (n=38) were stained for detection of nuclear pSTAT3 expression. Using a threshold of ≥30% of tumor nuclei staining positive, 35 % (14/40) of tumors were pSTAT3+. An additional 17% (7/40) had between 10–30% pSTAT3+ cells. Non-malignant tonsil tissues (n=10) were positive for tSTAT3 in all cases but all but one case were pSTAT3 negative. Twenty-four of the same DLBCL tumors used for pSTAT3 expression were stained for MYC and 50% (12/24) were MYC positive (all nuclear) as defined by the criteria of ≥30% of cells staining positive. In cases (n=23) where both MYC and pSTAT3 IHC were performed, a positive pSTAT3 was more likely to have MYC expression whereas a positive MYC stain did not inform pSTAT3. By using a break apart probe for the MYC gene, MYC translocations in the major breakpoint regions were found in 10% (3/29) of cases. When MYC FISH was correlated with MYC IHC in the 24 DLBCL cases that had both techniques performed, all 3 MYC translocation cases were GCB by IHC, two were strongly positive for MYC by IHC, and 1 was negative. Among the 21 MYC FISH negative cases, 10 were MYC positive by IHC. These data suggest that MYC expression in lymphoma is not only controlled by genetic events such as translocations but also by other signaling pathways such as STAT3. pSTAT3 expression was correlated with an elevated serum LDH (p=0.0007). Neither MYC or pSTAT3 tumor expression correlated with other clinical or pathological features. Survival analysis revealed a trend toward shorter EFS for DLBCL patients whose tumors expressed MYC protein by IHC (p=0.2) or had a MYC translocation (p=0.09) by FISH; pSTAT3 expression status did not predict EFS (p=0.9). Within the GCB group 10 cases were MYC negative (10/17; 59%) and 7 cases were MYC positive (7/17; 41%). Among the non-GCB group 83% (5/6) were MYC positive. These data clearly suggest a trend of higher MYC positivity in the non-GCB DLBCL group (p=0.07). MYC positive cases had a clear trend of inferior EFS in both GCB (p=0.2) and in non-GCB (p=0.5) groups. The distribution of pSTAT3 (n=38) expression was also evaluated in the GCB (n=26) and non-GCB (n=12) DLBCL. In the GCB group 27% (7/26) were pSTAT3 positive compared to 58% (7/12) in the non-GCB group. Thus, there was a clear trend toward higher pSTAT3 positivity in non-GCB DLBCL tumors (p=0.06); however, pSTAT3 status did not correlate with EFS in either GCB or non-GCB DLBCL patients. In the present study we correlated pretreatment serum cytokines levels of these patients with pSTAT3 and MYC tumor cell expression. Out of the 30 cytokines tested, the only pre-treatment cytokines that were significantly correlated with pSTAT3 expression were IL-10 (p=0.05), G-CSF (p=0.03) and TNFa (p=0.04); none were correlated with MYC expression.Overall, our data provide evidence that over-expression of pSTAT3 and MYC is common in DLBCL. These biomarkers have potential as prognostic factors in the case of MYC and as a tool for selecting therapy for pSTAT3. MYC may be especially useful to further identify an adverse group of DLBCL patients within the otherwise favorable GCB tumor group. The availability of JAK/STAT and STAT-specific inhibitors provides the rationale to incorporate pSTAT3 staining in tumors from patients who are participating in these trials to learn if this biomarker can predict response Disclosures:No relevant conflicts of interest to declare.

Abstract 1672: Population prevalence of consensus translocations in follicular and mantle cell lymphomas

Abstract Chromosomal translocations t(14;18)(q32;q21) of the anti-apoptotic gene BCL2 and t(11;14)(q13;q32) of the cell cycle regulatory gene cyclin D1 are hallmark mutations of follicular lymphoma (FL) and mantle cell lymphoma (MCL), respectively, both driven by the enhancer of the immunoglobulin heavy chain gene cluster IGH. These same translocations have also been detected at low frequency in peripheral blood lymphocytes (PBL) of clinically normal individuals, but association with risk of subsequent lymphoma remains to be determined. We therefore evaluated consensus PCR assays for these translocations towards development of potential blood screening tests of lymphoma risk. Formalin-fixed, paraffin-embedded tumor specimens were retrieved from 62 FL and 17 MCL diagnosed during a large population-based cancer screening trial. Lymphoma DNA was isolated from scrapings off glass slides and assessed for quality and quantity by quantitative real-time PCR amplification of the single copy reference gene KRAS. Samples were divided into three aliquots and tested with consensus primers to detect translocations within either the Major Breakpoint Region (MBR) on chromosome 18 or the Major Translocation Cluster (MTC) on chromosome 11, as appropriate. For MBR-positive reactions, PCR fragment length was estimated by agarose gel electrophoresis. Overall, 21 (34%) of 62 FL and 7 (44%) of 16 MCL were PCR-positive for the consensus translocation sequences. Translocation detection was highly dependent upon testing an adequate quantity of DNA, with 21 (45%) PCR-positives among 47 FL samples of at least 500 diploid genome equivalents vs. 0 (0%) positives among the remaining 15 FL (p&amp;lt;.001). PCR fragment lengths were identical from replicate assays for all but one MBR-positive sample, which yielded two products differing by 30 base pairs. Within the FL group, PCR-positivity did not significantly differ among Working Formulation histologies, including 2 (15%) of 13 mixed small cleaved and large cell FL, 5 (36%) of 14 small cleaved cell FL, 6 (55%) of 11 large cell FL, 1 (100%) of 1 centroblastic lymphoma and 7 (30%) of 23 FL not otherwise specified. We confirmed that nearly half of FL and MCL with adequate samples in this population-based cohort study have consensus translocation sequences. Thus, translocation presence and/or frequency in stored pre-diagnostic PBL can be usefully examined for associations with risk of subsequent lymphoma. FL- and MCL-associated translocations in PBL are a potential target for cancer risk prediction and preventive intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1672. doi:1538-7445.AM2012-1672

Mechanism of Fragility at BCL2 Gene Minor Breakpoint Cluster Region during t(14;18) Chromosomal Translocation

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at the 3'-UTR of BCL2 gene or downstream and are mainly clustered in either the major breakpoint region or the minor breakpoint cluster region (mcr). The recombination activating gene (RAG) complex induces breaks at IgH locus of chromosome 14, whereas the mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time, we show that RAGs can nick mcr; however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg(2+) and Mn(2+) are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at the mcr are independent of nonamer, whereas a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG-dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggest a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans.

Fine structure of translocation breakpoints within the major breakpoint region in BCR-ABL1-positive leukemias

The chromosomal translocation t(9;22)(q34;q22), with expression of the BCR-ABL1 fusion gene is the cytogenetic and molecular hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). Basically two types of BCR-ABL1 chimeric mRNA transcripts have been observed: (1) e13a2/e14a2 transcripts in CML and ALL, resulting from chromosomal breaks in the major breakpoint cluster region (M-bcr) of the BCR gene and (2) e1a2 transcripts in ALL resulting from breaks in the minor breakpoint cluster region (m-bcr) of the BCR gene. To gain a better understanding of this molecular alteration, we developed a long-distance inverse PCR (LDI PCR) method for M-bcr breakpoint identification in BCR-ABL1-positive cases and were thus able to identify the chromosomal breakpoints within the M-bcr in 62 BCR-ABL1-positive samples. The corresponding reciprocal breakpoints were identified and molecularly characterized in 45 of these cases. In 2 samples, the breaks were located 5′ to the ABL1 locus and in one case, the der(9) break was identified on 9q34.13 several hundred kB 3′ telomeric to ABL1. The analysis of breaks revealed no significant clustering and no association with repetitive elements (Alu, L1, L2) or recombination signal sequence sites. The established LDI PCR permits fast, relatively easy and unbiased identification of breakpoints in the M-bcr region of BCR and also enables the molecular analysis of more complex translocations with breakpoints outside the ABL1 gene locus or other BCR fusion genes.

Reply to V. Procházka et al

In their letter, Prochazka et al raise several issues as to the validity of our conclusion that in patients with relapsed or refractory follicular lymphoma, the presence of tumor cells in blood or bone marrow—as detected by BCL2/IgH major break point region polymerase chain reaction (PCR) at the end of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone induction therapy—is not predictive of progression-free survival (PFS). First, they suggest that we should have analyzed separately patients with clinical complete (CR) and partial remission (PR). We agree that this would have been a relevant subgroup analysis. Unfortunately, the four subgroups (PCRpositive CR, PCR-negative CR, PCR-positive PR, and PCR-negative PR) were too small to allow for a meaningful statistical comparison. However, as mentioned in our report, there was no difference between patients in clinical CR or PR with regard to the proportion of bone marrow (BM) or peripheral blood PCR negativity at the end of induction treatment (50% v 55%). This is clearly indicative of the compartmentalization effect and at least partly explains the lack of prognostic value of PCR status post induction treatment. Second, the authors state that our finding that PFS was not different between patients who converted to PCR negativity and those who did not is in contrast with previous publications by others. We agree and consider this to be an important message of our report. Prochazka et al mention two observations in our article that they consider to be supportive of the predictive value of PCR status. Their first consideration is based on our finding that there was highly significant improved PFS by rituximab maintenance in patients with postinduction PCR-negative BM, whereas the improvement was not significant in patients with PCR-positive BM (Figs 4A, 4B). In fact, there was a clear difference in the PCR-positive BM group, but this did not reach statistical significance because of limited numbers. However, highly significant improvement in PFS by rituximab maintenance was observed in both patients with postinduction PCR-negative blood and those with postinduction PCR-positive blood (Figs 4C, 4D). These observations do not change our conclusion that benefit from rituximab maintenance is independent of postinduction PCR status. Second, they mention our observation that patients who were still PCR positive at the end of maintenance treatment progressed rapidly after stopping maintenance therapy. In our opinion, this observation is not indicative of the predictive value of postinduction PCR status, but it is a strong argument for prolonging maintenance treatment.