Newly-diagnosed Diffuse Large B-cell Lymphoma Research Articles

Robust Assessment of Genomic Imbalance in Diffuse Large B-Cell Lymphoma Confirms Inferior Outcome Is Associated with Genomic Complexity and Identifies Potential Therapeutic Pathway Targets

Despite excellent initial responses of diffuse large B-cell lymphoma (DLBCL) patients to current frontline immunotherapy (RCHOP), only about 40% of patients are ultimately cured, with most relapses occurring within the first 2-3 years. Apart from clinical features, very few biomarkers are available and validated for risk stratification of DLBCL patients, other than cell-of-origin (COO) subtyping and MYC rearrangement. Genomic copy number changes identified using a variety of genomic profiling technologies including massively parallel sequencing, have variously been reported to have prognostic value in DLBCL. Our previous studies used a common analytical approach across three different clinical datasets to identify and define 32 common regions (CR) of overlapping genomic imbalance (17 gain, 15 loss), comprising 36 minimal common regions (MCR) observed in newly-diagnosed DLBCL. We now report the establishment of standardized calling criteria for a total of 50 aberrations within the 32 CR, taking into consideration GISTIC-defined peaks based on copy number data from three publicly available datasets: IS-172 (GSE11318, n=170), IS-51HR (E-MEXP-3463, n=51 with high IPI), and one similarly analyzed dataset IS-180 (GSE34171, n=180). As a form of validation of the scoring criteria, the frequencies of the aberrations detected in IS-172 in the known GCB and ABC COO subtypes were compared with those previously reported (PMID:18765795). The relative imbalance of the aberrations between the subtypes were re-capitulated including six occurring at higher frequency in the GCB subtype and five in the ABC subtype.Integration of the expression profiles of 162 samples of IS-172 by univariate t-test with the 50 aberrations revealed 569 unique positively-correlated Refseqs mapping to 24 of the MCRs with at least 1.2 fold change in expression (Univariate t-test with P ≤ 0.05 after Benjamini-Hochberg false discovery rate [FDR]). Of these, 27 were located within overlapping peaks of gain/loss. Ingenuity Pathway Analysis determined five significant pathways (P ≤ 0.001, FDR<0.10): p53 signaling, PKC theta signaling in T lymphocytes, geranylgeranyl-diphosphate biosynthesis, B-cell receptor signaling, and RANK signaling in osteoclasts.Association of each of the defined 50 aberrations with overall survival (OS) was performed with the Kaplan-Meier method and log-rank statistic in three RCHOP datasets: IS-124 (GSE15127, n=124), a subset of 70 of IS-180 (IS-70), and a dataset of 41 for which array-CGH was performed in-house using a targeted oligonucleotide (Agilent Technologies) with DNA extracted from sections of formalin–fixed paraffin-embedded de novo DLBCL (IH-41). Ten aberrations significantly associated (P ≤0.05) with poor outcome in at least one of the datasets: gain of 12q (12q13 and 12q14), 16q24, 19q13, and loss of 2q24, 2 sites at 6q21, 8p22, 9p21, 15q15, and 17p13. Of these 10 aberrations, loss of 17p significantly associated with gain of 16q, and losses of 6q, 8p, and 15q (P <0.05), while loss of 9p21 correlated with loss of 2q, 6q, and 8p as evaluated using the exact t-test. Gain of the 2 sites on 12q did not significantly correlate with any of the other 8 aberrations.Genomic complexity was assessed by two methods. In the first, the median number of MCR aberrations was determined to be 1, across three datasets (IS-172, IS-180, and IS-124). Specimens in RCHOP-treated datasets IS-124 and IS-70 were then called “complex” if 2 or more MCR aberrations were detected: 31% and 67% respectively. In both datasets, a complex genome was significantly associated with overall unfavorable outcome (P=0.037, 0.006 respectively). Specimens in IS-172 and IS-70 were additionally classified as “complex” based on the presence of at least one of nine aberrations involving the CDKN2A-TP53-RB-E2F axis (PMID:22975378) where 33% and 57% of cases were scored as complex respectively. Using this approach, significant association with outcome was only found for IS-70 (P<0.001). Of note, between “complex” scoring approaches, 11% and 13% discordance was evident.In summary, establishment of robust scoring criteria for copy number changes afforded correlative analyses with clinical features to reveal that genomic complexity is indeed associated with overall inferior survival in DLBCL and that integrated expression analysis identified biological pathways in DLBCL that may represent potential therapeutic targets. DisclosuresDias:Cancer Genetics,Inc: Employment. Thodima:Cancer Genetics,Inc: Employment, Stock option holder Other. Guttapalli:Cancer Genetics, Inc: Employment, Stock option holder Other. Mendiratta:Cancer Genetics,Inc: Employment. Houldsworth:Cancer Genetics, Inc.: Employment, Stock and stock option holder Other.

Quantitative Defects Of Cellular Immune Responses In Patients With Newly-Diagnosed Diffuse Large B-Cell Lymphoma

Altered immune status in non-Hodgkin lymphoma is characterized by undesired autoimmune phenomena and frequent occurrence of infections. The immune defects may be responsible for the initiation, maintenance and progression of malignant clone. We therefore evaluated immune effector cells between 35 patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) and 34 age- and sex- matched healthy controls using three-color flow cytometry. For patients with DLBCL, there were 19 males and 16 females with a mean age of 59.9 ± 15.6 years. There were 6, 17 and 13 patients with ECOG &gt; 2, stage III-IV and high intermediate to high International Prognostic Index, respectively. We found significant decreases in CD3+CD4+, CD4/CD8 ratio, CD4 central memory (CD4+CD45RO++CD27+) and B cells (Figure 1). From conditional logistic regression, only CD4/CD8 ratio showed significant association with being cases with the odds ratio of 0.29 (95% CI 0.12, 0.68). Disclosures: No relevant conflicts of interest to declare.

The Efficacy and Safety of the Addition of Rituximab to CHOP or a CHOP-Like Regimen In First Line Therapy for Diffuse Large B-Cell Lymphoma (DLBCL): A Meta-Analysis

AbstractAbstract 4913 Background:The addition of rituximab to cytotoxic chemotherapy has become standard in the initial treatment of DLBCL. However, estimations of the overall effect of the addition of rituximab to chemotherapy vary widely in the published literature. To date, no meta-analyses of rituximab in DLBCL have been published. We performed a comprehensive systematic review to compare chemotherapy with rituximab-chemotherapy in studies of newly-diagnosed DLBCL. The primary endpoint was OS; additional endpoints included disease control (DC), complete response (CR), and regimen-related toxicity (RRT). Disease control was assessed in each study as the time to treatment failure, event-free survival, progression free survival or time to progression. Methods:A comprehensive search for randomized trials (RCTs) comparing the addition of rituximab to chemotherapy was conducted in MEDLINE (January 1997 through April 2010). Also, meeting proceedings from American Society of Clinical Oncology, American Society of Hematology, European Society of Clinical Oncology and European Hematology Association, and studies listed on www.clinicaltrials.gov were manually searched for any supplementary abstracts, presentations or updates that were not identified in the original database search.The analysis included only RCTs comparing rituximab-chemotherapy with chemotherapy alone in patients with newly diagnosed DLBCL. Chemotherapy regimens were limited to CHOP or CHOP-like regimens. All three authors independently assessed each study's quality and performed blinded data extraction with conflict resolution by majority consensus. Given the inclusion of studies with both young and elderly patients, as well as HIV seropositive patients, we pooled data using a random effects model, with the estimate of heterogeneity being taken from the inverse variance fixed effect model. Results:Overall, seven RCTs involving 3539 patients with newly diagnosed DLBCL met inclusion criteria and were included in the meta-analysis. All studies were published as full-text articles. None of the studies were blinded. Studies from North America, Central America, and Europe were incorporated. The funnel plot for OS and Egger's test (1.37; 95% CI -2.25 to 4.99) did not indicate a significant publication bias. The test of heterogeneity among all RCTs was statistically significant in all endpoints. The pooled odds ratio (OR) demonstrated an increased OS for patients treated with rituximab-chemotherapy compared to chemotherapy alone (OR 0.71; 95% CI: 0.57 to 0.89). Similar improvements in CR (OR 1.64; 95% CI: 1.23, 2.17) and DC (OR 0.56; 95% CI: 0.46, 0.70) were observed in the patients treated with the rituximab containing regimens. Due to variable RRT reporting across studies, a formal meta-analysis of toxicity was not completed. In addition, varied data reporting precluded formal analysis of any differences in benefit between the International Prognostic Index groups. Conclusions:Our meta-analysis of data from more than 3500 patients with newly diagnosed DLBCL demonstrates that the addition of rituximab to chemotherapy improves the odds of OS and DC by 29% and 44%, respectively, as compared to chemotherapy alone. The survival benefit was independent of age (data not shown). This comprehensive systematic analysis enumerates and confirms the benefit of rituximab-based therapy in previously untreated DLBCL. [Display omitted] Forest Plot for OS. Disclosures:No relevant conflicts of interest to declare.

Incidence, Method and Covariates of Central Nervous System (CNS) Prophylaxis for Diffuse Large B-Cell Lymphoma In the National Comprehensive Cancer Network (NCCN) Lymphoma Database

AbstractAbstract 1756 Background:The optimal criteria to determine which patients with diffuse large B-cell lymphoma (DLBCL) should receive central nervous system (CNS) prophylaxis remain controversial. We aimed to characterize patterns of CNS prophylaxis administration in patients with DLBCL in a large multi-institutional database. Methods:The National Comprehensive Cancer Network (NCCN) Non-Hodgkin's Lymphoma Outcomes Database is a prospective cohort study collecting clinical, treatment, and outcomes data for patients at seven participating NCCN centers. Patients who presented between January 1, 2001 and July 1, 2008 with newly-diagnosed DLBCL, without CNS disease at baseline, and who received R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) within 180 days of diagnosis were eligible for this analysis. We assessed clinical and sociodemographic covariates of receipt of CNS prophylaxis and type of prophylaxis received (either ≥2 doses of intrathecal methotrexate and/or cytarabine or ≥1 dose of systemic methotrexate). Extrapolating from prior studies (e.g., Boehme et al, Ann Oncol, 2007), we defined high-risk features as >1 extranodal site involvement, elevated lactate dehydrogenase (LDH), bone marrow involvement, or other high-risk site involvement (orbit, testis, peripheral blood, bone/vertebrae, nasal/paranasal sinuses), and assessed rates of CNS prophylaxis in patients with ≥1 high-risk feature. We also determined rates of CNS recurrence for patients with ≥ 2 high-risk features who received prophylaxis versus those who did not. Results:Of a total of 989 patients with DLBCL (mean age 56; 44.4 % female; 70.2% with low or low-intermediate international prognostic index (IPI) score), 117 received CNS prophylaxis (11.8% [95% Confidence Interval (CI) 9.8%-13.8%]). Considered individually, patients with bone marrow involvement, other high-risk site involvement, >1 extranodal site involvement, high versus low IPI score, and advanced stage (III or IV) were significantly more likely to receive CNS prophylaxis (all p<0.0001); those with elevated LDH or presence of B-symptoms were not. In addition, age, gender, race/ethnicity, performance status, and cancer center were not associated with CNS prophylaxis receipt. Rates of CNS prophylaxis, in descending order of frequency, are shown below for significant clinical factors:Individual Clinical Factor+ CNS prophylaxisOther high-risk site involvement36.0%Bone marrow involvement28.2%>1 Extranodal site involvement23.1%High IPI18.6%Stage III/IV15.8%Of those who received CNS prophylaxis, 71.8% received intrathecal therapy and 28.2% received systemic therapy. There were significant center-specific differences regarding type of CNS prophylaxis used (p<0.0001). In addition, other high-risk site involvement was associated with use of systemic versus intrathecal prophylaxis (p=0.04), while higher mean age was associated with the reverse (p=0.05). Patients with increasing number of the four specific high-risk features (>1 extranodal site involvement, elevated LDH, bone marrow involvement, or other high-risk site involvement) were increasingly likely to receive CNS prophylaxis (0 features: 4.3%; 1 feature: 8.1%; 2 features: 20.1%; 3 features: 31.0%; 4 features: 61.1%; p<0.0001). Of all patients with ≥1 high-risk feature (n= 617), 16.4% received CNS prophylaxis. With median follow-up of 2.5 years, there were 20 CNS recurrences (2.0% [95% CI 1.1%-2.9%]). For those with ≥2 high-risk features (n=284), rates of CNS relapse were not significantly different in those who did and did not receive prophylaxis. Conclusion:In our cohort, several classic clinical high-risk variables were associated with receipt of CNS prophylaxis. The modality of prophylaxis used, whether intrathecal or systemic, appeared predominantly mediated by site-specific clinician preference rather than clinical or sociodemographic factors. While an increasing number of widely-accepted high-risk features was associated with higher rates of CNS prophylaxis, surprisingly, a minority of patients with high-risk features received CNS prophylaxis. In conclusion, we found no standard of care for administration of CNS prophylaxis in DLBCL; moreover, as the rate of CNS recurrence was low, our data suggest the need for further evaluation of the efficacy of CNS prophylaxis in the R-CHOP era. Disclosures:No relevant conflicts of interest to declare.

Variations in Surveillance Imaging for Patients with Treated Diffuse Large B-Cell Lymphoma in the National Comprehensive Cancer Network (NCCN) Lymphoma Database.

Abstract 1387Poster Board I-409 BACKGROUND:Utility and optimal timing of surveillance imaging for patients with treated diffuse large B-cell lymphoma (DLBCL) remains uncertain, especially in the era of positron emission tomography. METHODS:Data were obtained from the NCCN Non-Hodgkin's Lymphoma Outcomes Database, a prospective cohort study collecting comprehensive clinical, treatment, and outcome data for patients seen at 7 participating NCCN centers. Patients who presented between January 1, 2001 and June 30, 2007 with a new diagnosis of DLBCL in remission for at least three months after the completion of initial therapy were eligible for this study. For those patients with at least two years of follow-up, we assessed overall number and modality of imaging studies performed starting 90 days after the end date of their first-line therapy until documented relapse or end of two-year follow-up window. We then created three subcategories depending upon surveillance strategy: “PET” = positron emission tomography scans or combined positron emission tomography/limited computed tomography scans only; “CT”= computed tomography scans only; or “BOTH PET AND CT” = combination of both “PET” and “CT” at different times. For those who relapsed after being in primary remission for at least 90 days, we assessed the nature of the visit where relapse was first documented, recent accompanying imaging, and the presence of a confirmatory biopsy within one month before or after that relapse. RESULTS:As of July 1, 2009, 433 newly-diagnosed DLBCL patients had at least two years of follow-up from 6 of the 7 participating NCCN centers; median age was 56 and 57% were male. Initial stage and International Prognostic Index (IPI) were as follows: I = 24%; II= 24%; III= 16%; IV=36%; and low= 47%; low-intermediate= 26%; high-intermediate= 19% and high= 8%. For surveillance imaging as defined above, 14% received PET, 48% received CT, and 38% received BOTH PET AND CT. Range of median frequencies across NCCN centers are reported below, both overall and by test type. Overall median number of tests received per year differed significantly across NCCN centers (p for Kruskal-Wallis < 0.0001).Surveillance StrategyMedian tests/year across all NCCN centersMedian tests/year, NCCN center rangeMean tests/year across all NCCN centersAll tests2.81.5 – 3.52.8PET2.01.5 – 2.752.2CT2.51.5 – 3.52.6BOTH PET AND CT3.01.5 – 4.03.3Of 990 patients in primary remission for at least 90 days, there were 108 (11%) documented relapses. Of those, 20% were found at non-routine visits with symptoms, 41% at routine visits with symptoms, 37% at routine visits without symptoms and 2% were unclear. Of those found at routine visits without symptoms, 73% had undergone imaging within one month before and up to the date of relapse. Finally, 77% of those with relapse underwent confirmatory biopsy within one month before or after relapse. CONCLUSIONS:The frequency of surveillance imaging for patients with treated DLBCL is highly variable, even within the context of tertiary care academic centers. Despite controversy surrounding its use, a high proportion of DLBCL patients in remission are followed with PET modalities. Those being followed exclusively with PET modalities tend to have less frequent imaging, which may have implications for comparative effectiveness. Routine imaging likely identifies relapse in about a quarter of patients with DLBCL; the impact on ultimate outcome remains to be further evaluated. Disclosures:Friedberg: Genentech: Membership on an entity's Board of Directors or advisory committees. Blayney: American Society of Clinical Oncology: Membership on an entity's Board of Directors or advisory committees; BlueCross Blue Shield of Michigan: Research Funding; NCCN: Honoraria; University of Michigan Health System: Employment.

Who should receive myeloablative therapy for diffuse large B-cell lymphoma?

Over half of all newly-diagnosed diffuse large B-cell lymphoma patients can be cured with first line treatment based on the combination of rituximab with anthracycline-based chemotherapy regimens. The authors of this Viewpoint discuss the use of high-dose therapy and autologous hematopoietic stem cell support in patients with DLBCL who have relapsed or are refractory to first line treatment.

CHOP-R + bortezomib as initial therapy for diffuse large B-cell lymphoma (DLBCL)

8031 Background: Bortezomib is a proteasome inhibitor with anti-tumor activity in B cell malignancies. These effects, which may relate to NF-kappaB associated pathways, could sensitize tumor cells to standard chemotherapy-based regimens and enhance efficacy. We report findings of a phase I/II trial of dose-escalated bortezomib + standard CHOP-rituximab in DLBCL patients (accrual of the MCL cohort of this study remains ongoing). Methods: Patients with previously untreated DLBCL (n=40) received CHOP-21 + rituximab (375 mg/m2 each cycle) plus bortezomib at 0.7 mg/m2 (Arm 0, n=4), 1.0 mg/m2 (Arm 1, n=8) or 1.3 mg/m2 (Arm 2, n=28 including phase I and all phase II) on days 1 and 4 of each cycle Results: Median age (n=40) was 58 years (range 21–86), thirty-five subjects (88%) had stage III/IV disease at study entry, and 29 (73%) had elevated serum lactate dehydrogenase (LDH). Patients generally had unfavorable baseline international prognostic index (IPI) scores of 2 in 16 subjects (40%) and 3–5 in 19 subjects (48%). Median follow-up is 21 months (range 9 - 35 months). Treatment was generally well tolerated. Peripheral neuropathy occurred in 22 subjects (55%), with 45% grade 1, 5% grade 2 and 5% grade 3. Grade 4 hematologic toxicity included thrombocytopenia (15%) and leukopenia (15%). Four subjects (3 over age 75 and all with high risk IPI) died prior to first response assessment. Intent to treat (ITT) overall response rate (n=40) is 90% with 68% CR/CRu. For the evaluable subset (n=36), ORR was 100% with CR/CRu 75%. Kaplan-Meier estimate (n=40) of 2-year progression-free survival is 72%. Of all 19 enrolled (ITT) patients in the high-intermediate or high-risk IPI groups, 14 (74%) were alive without progression at last assessment. Correlation of outcome with cell of origin type (activated B cell vs germinal center) is ongoing. Conclusions: Bortezomib can be administered with acceptable toxicity in conjunction with CHOP-R chemotherapy. Efficacy findings with this combination regimen in newly-diagnosed DLBCL are encouraging and warrant further study. No significant financial relationships to disclose.

The addition of epratuzumab* and rituximab to CHOP** is effective for patients with newly-diagnosed diffuse large B-cell lymphoma

The addition of epratuzumab* and rituximab to CHOP** is effective for patients with newly-diagnosed diffuse large B-cell lymphoma

Rituximab, Bevacizumab (Avastin) and CHOP (RA-CHOP) for Patients with Diffuse Large B-Cell Lymphoma (DLBCL).

IntroductionBevacizumab is a humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF). Non-Hodgkin's lymphoma (NHL) patients with a high serum VEGF levels have an inferior overall survival compared to patients with low VEGF levels. Rituximab plus CHOP (R-CHOP) prolongs time to treatment failure and survival in patients with DLBCL. But further improvement is needed, particularly with patients with high-intermediate and high risk international index disease. This study was designed to assess the feasibility of the combination of bevacizumab plus R-CHOP (RA-CHOP).MethodBevacizumab was administered at 15 mg/kg IV on day 1 of each cycle over 30–90 minutes. R-CHOP was administered on day 2 of cycle 1 and on day 1 of subsequent cycles. Patients were monitored for cardiac function with echocardiogram and for proteinuria. Serum levels of bevacizumab and rituximab were measured to assess pharmacokinetics. Serum and urine samples were also analyzed for circulating VEGF. Tumor samples were stained for VEGF by immunohistochemistry.ResultsTwelve patients (10 males, and 2 females) with newly-diagnosed DLBCL were enrolled. The median age was 48 years (range 29–63). Three patients had stage II primary mediastinal, 2 stage III, and 7 stage IV disease. ECOG performance status ranged from 0–1. Nine patients had an elevated LDH with a median international index score of 2 (range 0–3). Nine patients had an international index score of 2–3. A total of 80 cycles (range 2–8; median 7) of RA-CHOP was administered. Bevacizumab was held in 5 cycles in 3 patients-due to 1) elevated liver enzymes in the setting of positive hepatitis C serology, 2) transient proteinuria, and 3) DVT associated with central line. The following grade 3 toxicities were observed: 4 febrile neutropenia with infection related to central line, 2 DVT (both central line associated), 1 elevated liver enzymes, and 2 transient hypertension. Grade 4 toxicities included 3 absolute neutropenia with 2 febrile neutropenia, and 1 HSV esophagitis. Two patients are too early to assess response. Best response included CR in 3, PR in 4 (2 with normal PET scans), SD in 1. One patient progressed and another achieved a PR after 4 cycles but further treatment was not given due to severe depression.ConclusionIn this patient population, treatment with RA-CHOP did not result in any grade 3 or 4 proteinuria, heart failure or bleeding. Hypertension was transient and thrombosis was associated with central line placement. Correlative studies and pharmacokinetics are being analyzed and will be available for presentation.

Identification of Genetic and Expression Markers in DLBCL Cell Lines That Influence the In Vivo Response of DLBCL to Doxorubicin.

Diffuse large B-cell lymphoma (DLBCL) is the largest aggressive group of non-Hodgkin's lymphoma (~40%) and exhibits great clinical and pathological heterogeneity. While DLBCL represents a chemosensitive disease, ultimately, only approximately 50% of patients are cured with current treatment options. Standard front-line therapy comprises CHOP and more recently Rituximab-CHOP. CHOP commonly contains doxorubicin as the curative anthracycline. Controversy currently exists in the use of genetic and expression markers in predicting response to treatment and outcome of DLBCL, thus, we examined the cellular responses of a panel of eight DLBCL cell lines to doxorubicin in order to define molecular markers that influence the response. Expression profiling and subsequent classification of the cell lines revealed that six of the lines were of a germinal center B-cell-like (GCBL) subtype and two (Ly3 and Ly10) were of a non-GCBL subtype. Ly3 and Ly10 exhibited a rapid apoptotic response within 24 hours after exposure to doxorubicin as evaluated by Hoescht staining. Expression profiling of the response of Ly10 cells (in triplicate) revealed the up-regulation of transcripts consistent with mediation of the cellular response via a p53-dependent pathway. Mutation analysis and Western blotting revealed wild type TP53 in Ly3 and Ly10 with an induction of p53 levels after doxorubicin. The remaining cell lines exhibited mutated TP53 sequences and no induction of p53 by doxorubicin. Therefore, in DLBCL cell lines, wild type p53 effects an early rapid apoptotic response to doxorubicin. In the six non-GCBL cell lines, a decrease in viability after doxorubicin was observed at much later times (up to 48 hours after treatment) by both apoptotic (exemplified by Ly2 and Ly8) and non-apoptotic mechanisms (exemplified by Ly1). Expression profiling of these three cell lines revealed alterations in expression of transcripts consistent with a G2/M cell cycle delay induced by doxorubicin, which was confirmed by follow-up flow cytometric analysis. An induction of transcripts consistent with NF-κB and AP-1 activation was observed in Ly1, but not in Ly2 and Ly8, which was confirmed by electrophoretic mobility shift assays. In order to determine if this cellular response plays a role in determining the response of DLBCL to doxorubicin in vivo, the expression profiles of untreated Ly1 and Ly2 were compared, and the resulting 160 differentially expressed transcripts were used to cluster 41 newly-diagnosed DLBCL specimens which had previously been profiled and had a known response to doxorubicin-containing therapy. This analysis revealed a marked correlation between capacity to induce NF-κB and AP-1 with in vivo treatment failure. Overall, these experimental studies have begun to outline genetic and biologic features that together contribute to the response of DLBCL to doxorubicin in vivo.

Clinical Outcome Correlations of Genomic Gains and Losses in Diffuse Large B-Cell Lymphoma by Array-CGH.

Conventional karyotype and chromosomal comparative genomic hybridization (CGH) studies of diffuse large B-cell lymphoma (DLBCL) have revealed few chromosomal abnormalities associated with outcome. In order to evaluate the association between genomic copy number changes in DLBCL and clinical outcome at a higher resolution, we assayed a panel of 64 newly-diagnosed DLBCL specimens by array-CGH. For each specimen, the respective patient had a known response to anthracycline-based therapy, and the median follow-up was 5-years. Forward and reverse hybridizations were performed for all specimens to avoid dye-bias, using BAC/PAC arrays with 1-4 Mb resolution coverage of the genome (Spectral Genomics). After customized normalization, the circular binary segmentation (CBS) algorithm was used to identify regional copy number changes along each chromosome in each specimen. Of the 64 specimens, 59 (92%) displayed regional copy number changes. The frequency of gain/loss of each clone for all 64 specimens was then calculated based on the CBS results, and, additionally, based on singleton clone changes if they were outside of CBS-defined regions. Clones displaying change ≥ in 10% of specimens for at least two contiguous clones were further considered as recurrent sites for clinical correlations. 27 sites of recurrent gain and 25 sites of recurrent loss were identified. Association between loss or gain of sites and International Prognostic Index (IPI) were evaluated using Fisher's exact test, and associations between loss or gain of sites and time to treatment failure (TTF) and overall survival (OS) were tested using the log-rank test. Table 1 lists the sites as independent markers that were significant at p < 0.05. Of these sites, five (with asterisk) were found to significantly predict outcome after stratifying by IPI using the stratified log-rank test. It is noted that 6 of the sites were 5 Mb or less in size, facilitating the identification of target genes. Losses of chromosome 7 (8–13 Mb) and chromosome 13 (56.7–63 Mb) were found to contribute to IPI prediction of outcome, but were not significantly associated with clinical features as independent markers. In summary, array-CGH has lead to the identification of several genetic markers with prognostic significance in DLBCL, which in some cases are of a size amenable for target gene identification.Significant Genetic Markers Associated with Clinical OutcomeChrom.Start (Mb)End (MB)Change# of casesIPITTFOS178.279.1Loss19Better22.44.1Loss8Worse*30.272.3Gain14BetterBetter3101.3136Gain18LowBetter3154.7188.7Gain18LowBetterBetter662.2170.5Loss32LowBetterBetter718.819.2Loss11Better*98.313Loss13Better983.996.3Gain9Better9122.1132.8Gain12BetterBetter*120133.3Gain31High160.14.7Gain13Better1633.835.6Loss7Worse*Worse*1943.163.7Gain13BetterBetter