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Background With increased understanding of the unique entities, subtype-specific approaches for peripheral T-cell lymphoma (PTCL) are emerging, and more precise diagnoses are becoming increasingly important. Patients and Methods We analyzed the approach to the histopathologic diagnosis of PTCL using data from the comprehensive oncology measures of peripheral T-cell lymphoma (COMPLETE) study. The COMPLETE trial is a large prospective cohort study of patients with newly diagnosed PTCL in the United States. Results A total of 499 patients were enrolled from 40 academic and 15 community-based centers. Baseline assessment forms were collected for 493 patients, of which 435 (88%) were available for analysis. The most common diagnoses were PTCL, not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, and angioimmunoblastic T-cell lymphoma (AITL). A mean of 10 markers (range, 0-21) was assessed per patient. CD30 was assessed frequently but not uniformly in cases that were not anaplastic large cell lymphoma. Only 17% of PTCL-NOS cases were assessed for PD1. CXCL13 was a relatively sensitive marker in AITL, expressed in 84% of tested cases; however, only 3% of PTCL-NOS cases were tested. T follicular helper cell marker assessment differed between academic and community practices, with PD1 more often evaluated by academic centers in cases of AITL (62% vs. 12%; P = .01). Conclusion The diagnostic workup for PTCL in the United States varies widely and often lacks important phenotypic information to fully characterize the lymphoma. Gaps in testing of selected markers should be filled, given the impending revision to the World Health Organization classification. The accuracy of diagnosis will become increasingly important as we enter the era of targeted treatment for PTCL.

Diffuse large B-cell lymphoma (DLBCL) is a clinically and pathologically heterogeneous entity. Multiple molecular methods have been used to further understand and define this heterogeneity. Gene profiling has identified “cell-of-origin” molecular subtypes of DLBCL based on expression pattern

Myeloid sarcoma manifesting in the testis is rare and may occur concomitantly with bone marrow disease or as a separate entity. We describe our experience with a 6-month-old boy who presented with painless scrotal swelling and was found to have bilateral testicular masses on ultrasonography. The patient underwent unilateral radical inguinal orchiectomy. Surgical pathology revealed myeloid sarcoma of the testicle. He developed peripheral blood involvement 1 week postoperatively. Bone marrow biopsy showed acute myeloid leukemia. He is in remission after 2 cycles of induction chemotherapy, local radiation therapy, and allogeneic bone marrow transplantation.

Melanoma accounts for most skin cancer-related deaths and has an increasing incidence. Accurate diagnosis and distinction from atypical nevi can be at times difficult using light microscopy alone. Fluorescence in situ hybridization (FISH) and melanoma gene expression score (myPath, Myriad Genetics) have emerged as ancillary tools to further aid in this differential diagnosis. Our aim in this study was to correlate FISH results, gene expression score, consensus histopathologic impression and clinical outcome on a series of 117 challenging melanocytic lesions collected from three separate institutions. The lesions were separated into two groups: 39 histopathologically unequivocal lesions (15 malignant, 24 benign) and 78 challenging lesions interpreted by expert consensus (27 favor malignant, 30 favor benign, and 21 ambiguous). Melanoma-FISH was performed using probes for 6p25, 11q13, 8q24, and 9p21/CEP9 and scored according to established criteria. Analysis by myPath gene expression score was performed and interpreted by the manufacturer as 'benign', 'indeterminate,' or 'malignant'. In the unequivocal group, melanoma-FISH and myPath score showed 97 and 83% agreement with the histopathologic diagnosis, respectively, with 93 and 62% sensitivity, 100 and 95% specificity, and 80% inter-test agreement. In the challenging group, FISH and the myPath score showed 70 and 64% agreement with the histopathologic interpretation, respectively, with 70% inter-test agreement and similar sensitivities and specificities. The inter-test agreement was 73% overall, excluding indeterminate results. Discordant test results occurred in 27/117 cases from both unequivocal and challenging groups. Melanoma-FISH and gene expression score are valuable ancillary tools, though both have limitations and return discordant results in a subset of cases. Follow-up studies with more extensive clinical outcome data are warranted to establish the accuracy of these tests for the classification of melanocytic lesions.

Recent studies suggested that MYC and BCL2 protein co-expression is an independent indicator of poor prognosis in diffuse large B-cell lymphoma. However, the immunohistochemistry protocols for dual-expression staining and the scoring cut-offs vary by study. Sixty-nine cases of diffuse large B-cell lymphoma were evaluated for MYC and BCL2 protein expression using various cut-offs that have been recommended in prior studies. Independent of the International Prognostic Index risk group, cases with dual protein expression of BCL2 and MYC using ≥50%/40% cut-offs and ≥70%/40% had significantly shorter overall survival than cases without. It was verified in this patient population that the use of BCL2 and MYC immunohistochemistry, performed with available in vitro diagnostic-cleared antibodies, provides rapid prognostic information in patients with de novo diffuse large B-cell lymphoma. This study has practical implications for diagnostic laboratories and serves as a guide for implementation in the setting of future clinical trials.

To identify pretest characteristics of cerebrospinal fluid (CSF) specimens that will allow the rational use of flow cytometric analysis (FCA) in the diagnosis of hematologic malignancy.Retrospective data were collected on 501 consecutive CSF samples submitted for FCA.A positive diagnosis of hematologic malignancy was made in 41 specimens (8.2%). Blasts or atypical lymphocytes were noted on Wright-stained slides in 98% of FCA-positive specimens (40/41), and a history of a hematologic malignancy was present in 89% of specimens (34/38). All FCA-positive specimens had atypical morphology or history of hematologic malignancy. Four hundred six specimens (81%) were FCA negative. Of FCA-negative specimens, 7% (30/406) had atypical morphology, and 3% (12/404) had future central nervous system involvement seen within 30 days.These data support a policy in which FCA of CSF is actively discouraged unless atypical lymphocytes or blasts are seen or a history of hematologic malignancy is present.

Background Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease entity with multiple potential prognostic biomarkers. Cell of origin (COO) molecular subtype classification using gene expression profiling with microarrays and immunohistochemical expression of CD30 have been examined as potential prognostic markers, often with conflicting results. A recent study demonstrated that patients with CD30-positive DLBCL had better prognosis compared to patients with CD30-negative DLBCL and had a distinct gene expression profile (Hu S et al. Blood. 121(14): 2715-24, 2013). In addition, due to the development of targeted therapies such as an anti-CD30 monoclonal antibody drug conjugate, the identification and prognostic relevance of this biomarker has potential therapeutic impact. We evaluated CD30 expression, determined by immunohistochemistry, in a cohort of de novo DLBCL cases at our institution and examined cell of origin molecular subtype in the CD30-positive and CD30-negative groups. Design 94 adult patients with de novo DLBCL uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) as first line therapy were identified at The Cleveland Clinic. Clinical data was collected for these patients. A tissue microarray was created and stained with antibodies to CD10, CD20, CD30, BCL-6, and MUM-1. COO subtype was determined for the Hans algorithm in all cases. CD30 was considered positive when expression was seen in ≥ 20% of tumor cells. Results There were no significant differences in sex, age, IPI score, or stage between patients in the CD30-positive and CD30-negative groups. The median age of the DLBCL cohort was 63 years (range 17-91 years) with a male : female ratio of 1:1.1. 54% of patients had stage III or IV disease. Median follow-up was 58 months. 9 of 94 DLBCL samples (9.6%) were positive for CD30 by immunohistochemistry. By Kaplan-Meier analysis, the CD30-positive cases showed a decreased overall survival compared to the CD30-negative cases (Figure 1, p=0.044). Multivariate analysis using a Cox proportional hazard model confirmed that CD30 expression was independent of IPI and a significant factor for overall survival (hazard ratio = 3.05; 95% confidence interval = 1.12-8.30; p = 0.0291). All 9 of the CD30-positive DLBCL samples were of the non-germinal center B-cell-like (NGC) subtype using the Hans immunohistochemical algorithm, which was significantly more than the CD30-negative samples (42/85) (p = 0.003). Conclusion CD30 expression was associated with poor prognosis in our cohort, in contrast to recent studies. However, CD30 expression was highly associated with the NGC subtype of DLBCL and might contribute to the pathogenesis of these lymphomas through NF-κB activation. Given the poor prognosis of NGC DLBCLs, targeting CD30 in DLBCL should be explored. Disclosures: No relevant conflicts of interest to declare.

Abstract 950 INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) can be categorized by its cell of origin (CoO) as either being derived from a germinal center B-cell (GCB) or activated B-cell (ABC). Primary mediastinal DLBCL represents a third, distinct entity. This classification was initially defined by gene expression profiling (GEP), which remains the gold standard for such determination. Determination of CoO will likely become the basis for patient selection for clinical trials of targeted therapies. Several algorithms and methods have been developed that use immunohistochemistry (IHC) to differentiate GCB-DLBCL from non-GCB DLBCL. These include the Hans algorithm (utilizes staining for CD10, Bcl-6 and Mum1), the Choi algorithm (utilizes additional staining for GCET1 and FoxP1) as well as the Tally method (does not use Bcl-6 and utilizes LMO2 as a tie-breaker stain for otherwise equivocal cases). Recently, it has been recognized that IHC approaches to assign CoO may not be reproducible even at highly experienced laboratories. We sought to determine the performance of these IHC assays in our laboratory as a necessary step in developing trials based on CoO stratification. METHODS: We reviewed 108 adult (age ≥18) cases of de novo DLBCL, the majority of which were treated with chemoimmunotherapy (R-CHOP or R-CVP) at the Cleveland Clinic from 2000–2010. Diagnostic biopsies were available for all cases. IHC staining was performed on tissue microarrays (TMAs), and published algorithms (Hans, Choi and Tally) were applied to categorize cases as GCB or non-GCB. In addition, gene expression profiling was completed in a subset of these cases, for which frozen tissue was available. A linear predictor score for gene expression profiling (GEP) was used to assign cases in 31 of 33 cases with 2 technical failures at the array stage (overall success rate 84.8%). Clinical details including age, sex, International Prognostic Index (IPI) stage at diagnosis, treatment, progression free survival (PFS) and overall survival (OS) were captured for 69 of the 108 patients. Actuarial survival analysis was performed according to the Kaplan and Meier method, and the curves compared by the log-rank test. RESULTS: For the 69 patients with adequate clinical follow-up, the median age was 64 years old (range 18–88). There were 49% males and 51% females. The distribution of patients with stage I, II, III, and IV disease at the time of diagnosis was 20%, 14%, 20%, and 32% (14% had unknown stage). The 5-year overall survival of patients was 88%. Results of the Hans algorithm, Choi algorithm and Tally method were interpretable in 98 (90.7%), 95 (87.9%) and 88 (81.5%) of 108 cases, respectively. Inability to assign subtypes was due to suboptimal staining of the TMA (tissue loss or poor staining of an individual core). Using GEP to assign CoO, 42% of cases were classified as GCB, 42% as ABC and 14% were unclassifiable. The sensitivities of the Hans, Choi and Tally approaches to identify the CoO predicted by GEP were 0.83, 0.83, and 0.58 for correctly identifying GCB cases, respectively, and were 0.70, 0.70 and 0.80 for identifying non-GCB cases, respectively. The positive predictive values of the Hans, Choi and Tally approaches were 0.83, 0.83, and 1.0 for GCB and 0.78, 0.78, and 0.89 for non-GCB. As shown in the figure, 5-year overall survival was significantly superior for GCB relative to ABC cases using GEP (100% vs. 58.9%, P < 0.001) and for GCB vs. non-GCB cases for the algorithms of Hans (100% vs. 82.3%, P = 0.0197) and Choi (95.6% vs. 78.0%, P = 0.0482). The Tally method was not predictive of outcome, possibly due to insufficient power (5-year OS 94.4% for GCB vs. 80.7% for non-GCB, P = 0.1725). Similar findings were observed for progression-free survival. CONCLUSIONS: The Hans and Choi algorithms are reasonable methods for identifying PFS and OS differences based on CoO for de novo DLBCL treated with chemoimmunotherapy. The positive predictive value is universally high for all algorithms tested, but the sensitivity of IHC for identifying CoO was fair, particularly for the Tally method. IHC represents a valid biomarker to identify non-GCB cases. Clinical trials of DLBCL that stratify patients by IHC are feasible provided the performance characteristics of the algorithms are taken into consideration during study design. Disclosures: No relevant conflicts of interest to declare.

Introduction Diffuse large B cell lymphoma (DLBCL), the most common lymphoma world-wide, is strikingly heterogeneous. This heterogeneity creates a daunting challenge for conducting well-powered studies connecting molecular features to clinical outcome. Not only is the association of genetic mutations with clinical outcome in DLBCL mostly unknown, the relative importance of other well-described features, such as MYC and BCL2 translocation/expression and cell of origin based subsets (ABC and GCB DLBCL), is difficult to interpret due to conflicting reports. We sought to comprehensively define the spectrum of genetic mutations and their association with clinical outcome in DLBCL. Our calculations indicated that 500 tumor-normal pairs would provide 95% power to define mutations occurring in at least 5% of patients, and that 800 cases would be required to define the clinical correlations with cross-validation. Methods We enrolled 1001 de novo DLBCL patients, with complete IPI and survival data, who were treated uniformly with standard rituximab and anthracycline containing regimens. All tumors were subjected to whole exome and transcriptome sequencing (RNAseq), as well as SNP arrays to confirm genetic alterations. ABC (38%) and GCB DLBCL (36%) subtypes were defined using microarrays and RNAseq in these patients to examine subgroup-based differences in mutations and outcome. MYC and BCL2 expression were quantified separately. Results Gene discovery analysis of somatic mutations and copy number alterations in exome sequencing data from 502 tumor-normal pairs of DLBCL identified 197 recurrently mutated genes, including 155 genes previously identified to be mutated in DLBCLs. In addition, our study uncovered 42 novel driver genes in DLBCL (e.g. BTK, SPEN, CD70). Exome sequencing results were validated by Sanger sequencing of 1120 variants with over 90% concordance. We also identified copy number alterations in these genes, with strong agreement (90%) of amplifications and/or deletions to those detected on Illumina high resolution SNP microarrays. These 197 genes were found to comprise 15 functionally related subnetworks, including those related to histone modification, NFkB, B cell receptor, PI3K and cell cycle (Figure 1). Within each subnetwork, the vast majority of the gene alterations occurred in a mutually exclusive (P We examined the associations between the mutations and clinical outcome in all 1001 patients. All survival analyses were conducted using nearly equally split training and validation sets, corrected for multiple comparisons with significance of P Conclusion To our knowledge, this is the largest whole exome sequencing study in any single cancer. Our study answers many long-standing questions in the disease, informing a comprehensive understanding of genetic drivers of DLBCL, their organization into pathways, and their relationship to clinical outcome. Disclosures Leppä: Roche: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda Pharmaceuticals: Consultancy, Honoraria, Other: Travel expenses; Janssen: Consultancy, Research Funding; CTI Bio Pharma: Consultancy; Mundipharma: Research Funding; Bayer: Other: Travel expenses, Research Funding. Flowers:ECOG: Research Funding; Gilead: Consultancy, Research Funding; Millenium/Takeda: Research Funding; Pharmacyclics, LLC, an AbbVie Company: Research Funding; TG Therapeutics: Research Funding; Mayo Clinic: Research Funding; NIH: Research Funding; Infinity: Research Funding; AbbVie: Research Funding; Genentech: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Acerta: Research Funding. Hsi:Seattle Genetics: Honoraria, Speakers Bureau; HTG Molecular Diagnostics: Consultancy, Honoraria; Eli Lilly: Research Funding; Cellerant Therapeutics: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Evens:Takeda: Other: Advisory board. Reddy:GILEAD: Membership on an entity's Board of Directors or advisory committees; INFINITY: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees.