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Aims and methodsAccurate protein measurements using formalin-fixed biopsies are needed to improve disease characterisation. This feasibility study used targeted and global mass spectrometry (MS) to interrogate a spectrum of disease severities using 19 ulcerative colitis (UC) biopsies.ResultsTargeted assays for CD8, CD19, CD132 (interleukin-2 receptor subunit gamma/common cytokine receptor gamma chain), FOXP3 (forkhead box P3) and IL17RA (interleukin 17 receptor A) were successful; however, assays for IL17A (interleukin 17A), IL23 (p19) (interleukin 23, alpha subunit p19) and IL23R (interleukin 23 receptor) did not permit target detection. Global proteome analysis (4200 total proteins) was performed to identify pathways associated with UC progression. Positive correlation was observed between histological scores indicating active colitis and neutrophil-related measurements (R2=0.42–0.72); inverse relationships were detected with cell junction targets (R2=0.49–0.71) and β-catenin (R2=0.51–0.55) attributed to crypt disruption. An exploratory accuracy assessment with Geboes Score and Robarts Histopathology Index cut-offs produced sensitivities/specificities of 72.7%/75.0% and 100.0%/81.8%, respectively.ConclusionsPathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

Immunohistochemistry (IHC) using formalin-fixed, paraffin embedded (FFPE) tissue is limited by epitope masking, posttranslational modification and immunoreactivity loss that occurs in stored tissue by poorly characterized mechanisms. Conformational epitopes recognized by many programmed-death-ligand-1 (PD-L1) IHC assays are particularly susceptible to degradation and provide an ideal model for understanding signal loss in stored FFPE tissue. Here we assessed 1206 tissue sections to evaluate environmental factors impacting immunoreactivity loss. PD-L1 IHC using four antibodies (22C3, 28-8, E1L3N, and SP142), raised against intracellular and extracellular epitopes, was assessed in stored FFPE tissue alongside quantitative mass spectrometry (MS). Global proteome analyses were used to assess proteome-wide oxidation across an inventory of 3041 protein groups (24,737 distinct peptides). PD-L1 quantitation correlated well with IHC expression on unaged sections (R2 = 0.744; P

Introduction The hepatocyte growth factor receptor mesenchymal-epithelial transition (MET) is reported to be a negative prognostic marker in EGFR-mutant NSCLC and involved in resistance to EGFR inhibitors. Emibetuzumab, a humanized immunoglobulin G4 monoclonal bivalent MET antibody, blocks ligand-dependent and ligand-independent hepatocyte growth factor/MET signaling. This phase 2 study compared erlotinib with and without emibetuzumab in first-line treatment of EGFR-mutant metastatic NSCLC. Methods Patients with stage IV EGFR-mutant NSCLC and disease control after an 8-week lead-in with erlotinib (150 mg daily) were randomized to continue taking erlotinib with or without emibetuzumab (750 mg every 2 weeks). The primary end point was progression-free survival (PFS). Additional end points included overall survival, overall response rate, safety, pharmacokinetics, and exploratory analysis of MET expression. Results No significant difference in median PFS was observed in the intent-to-treat population (9.3 months with emibetuzumab + erlotinib versus 9.5 months with erlotinib monotherapy [hazard ratio (HR) = 0.89, 90% confidence interval (CI): 0.64–1.23]). The median overall survival was 34.3 months with emibetuzumab plus erlotinib versus 25.4 months with erlotinib (HR = 0.74, 90% CI: 0.49–1.11). Emibetuzumab plus erlotinib was well tolerated, with peripheral edema and mucositis as the only adverse events occurring 10% or more frequently relative to erlotinib. Exploratory post hoc analysis showed an improvement of 15.3 months in median PFS for the 24 patients with the highest MET expression (MET expression level of 3+ in ≥90% of tumor cells) (20.7 with emibetuzumab + erlotinib versus 5.4 months with erlotinib [HR = 0.39, 90% CI: 0.17–0.91]). Conclusions No statistically significant difference in PFS was noted in the intent-to-treat population. Exploratory analysis confirmed that high MET expression is a negative prognostic marker for patients treated with erlotinib, indicating that emibetuzumab plus erlotinib may provide clinically meaningful benefit.

Ectodomain shedding unleashes the aggressive nature of the MET oncogene product. Using specific C- and N-terminal MET antibodies (D1C2 and A2H2-3), MET protein status (i.e., no MET, decoy MET, transmembranous C-terminal MET with or without the ectodomain) was investigated in oral squamous cell carcinoma. For the cancers showing transmembranous C-terminal MET, the impact of ectodomain shedding on prognosis was investigated. To examine ectodomain shedding, reduced lysates of oral squamous cell carcinoma cell lines were immunoblotted using D1C2 and an ELISA was performed on culture media using A2H2-3. In addition, reduced lysates of fresh frozen tissues of 30 oral squamous cell carcinoma were immunoblotted using D1C2 and immunohistochemistry was performed on corresponding formalin-fixed paraffin-embedded tissues using both antibodies on parallel sections. To examine MET protein status, differences between membranous D1C2 and A2H2-3 immunoreactivities were scored using parallel tissue microarray sections representing 156 oral squamous cell carcinoma. The prognostic value of ectodomain shedding was examined using Cox regression analysis for disease-free survival and overall survival. Ectodomain shedding was observed in all cell lines, 43% (n = 13) of fresh frozen and 50% (n = 15) of formalin-fixed paraffin-embedded cancers (27% overlap, n = 8). The tissue microarray showed no MET in 23% (n = 36), decoy MET in 9% (n = 14), and transmembranous C-terminal MET in 68% (n = 106) of examined cancers. Within the latter group, ectodomain shedding occurs in 36% (n = 38) of the cases and is independently associated with poor disease-free survival (HR = 2.41; 95% CI, 1.35–4.30 and P = 0.003)—though not overall survival (HR = 1.64; 95% CI, 0.92–2.94 and P = 0.095)—after correcting for factors known to influence survival. In conclusion, MET ectodomain shedding occurs in transmembranous C-terminal MET positive oral squamous cell carcinoma and is independently associated with disease-free survival. These findings might aid in designing companion diagnostics for targeted therapies directed against MET.

Using tissue microarrays, it was shown that membranous C-terminal MET immunoreactivity and ectodomain (ECD) shedding are associated with poor prognosis in oral cancer. Seen the potential diagnostic value, extrapolation of these results to whole-tissue sections was investigated. Because MET orchestrates epithelial-to-mesenchymal transition (EMT), the results were benchmarked to loss of E-cadherin, a readout for EMT known to be associated with poor prognosis. C-terminal MET, N-terminal MET, and E-cadherin immunoreactivities were examined on formalin-fixed paraffin-embedded parallel sections of 203 oral cancers using antibody clones D1C2, A2H2-3, and NCH-38. Interantibody and intra-antibody relations were examined using a novel scoring system, nonparametric distribution, and median tests. Survival analyses were used to examine the prognostic value of the observed immunoreactivities. Assessment of the three clones revealed MET protein status (no, decoy, transmembranous C-terminal positive), ECD shedding, and EMT. For C-terminal MET-positive cancers, D1C2 immunoreactivity is independently associated with poor overall survival (hazard ratio [HR] = 2.40; 95% confidence interval [CI] = 1.25 to 4.61; and P = 0.008) and disease-free survival (HR = 1.83; 95% CI = 1.07-3.14; P = 0.027). For both survival measures, this is also the case for ECD shedding (43.4%, with HR = 2.30; 95% CI = 1.38 to 3.83; and P = 0.001 versus HR = 1.87; 95% CI = 1.19-2.92; P = 0.006) and loss of E-cadherin (55.3%, with HR = 2.21; 95% CI = 1.30 to 3.77; and P = 0.004 versus HR = 1.90; 95% CI = 1.20-3.01; P = 0.007). The developed scoring system accounts for MET protein status, ECD shedding, and EMT and is prognostically informative. These findings may contribute to development of companion diagnostics for MET-based targeted therapy.

The objective of this study was to measure concordance of results obtained from the US Food and Drug Administration-approved Ki-67 immunohistochemistry MIB-1 pharmDx assay performed on the Dako Omnis automated staining instrument (Omnis) versus results produced from the assay reagents applied using an optimized protocol on the more widely available Autostainer Link 48 (ASL48) platform. Tissue sections obtained from 40 formalin-fixed paraffin-embedded breast carcinoma samples, with available Oncotype DX Breast Recurrence Score (RS) results, were stained. Three certified pathologists scored slides at 3 timepoints, totaling 360 observations for each instrument (N=720 total) using the approved scoring approach. Using the ≥20% cutoff, agreement was calculated with corresponding 2-sided 95% percentile bootstrap confidence intervals (CIs). Pairwise comparisons (N=360) from the interinstrument evaluation, performed with all observers, resulted in 325 (90.3%) concordant outcomes (244 negative and 81 positive) and 35 (9.7%) discordant outcomes. The overall agreement was 90.3% (95% confidence interval, 85.6% to 94.4%). No significant systematic differences were observed between instruments. Specimens scored from the Omnis were on average1% higher than ASL48, with high correlation and little bias between the continuous Ki-67 scores (concordance correlation coefficient=0.916). Most specimens with a Ki-67 score ≥20% had a RS25. This study demonstrated that good concordance can be achieved with the reagents run on the ASL48 instrument when using an optimized protocol and standardized scoring.

Simple Summary Pancreatic cancer is one of the most lethal types of cancer, and immunotherapy treatment options for these patients are limited by the characteristically “cold” tumor microenvironment. In this work, we analyze the expression levels and prognostic value of stromal tumor-infiltrating lymphocyte (CD4, CD8, and CD20) and cancer-associated fibroblast (Thy-1, FAP, and SMA) subpopulations in a cohort of pancreatic ductal adenocarcinoma patients. We additionally characterize the expression and prognostic value of CD200, a potential target for immune checkpoint blockade in these patients. CD8 and FAP were found to have prognostic significance for progression-free survival and overall survival after multivariate analysis. CD200 expression was heterogeneous in tumor and stromal cells and did not demonstrate prognostic value in this cohort. Our results point to CD8 and FAP as potential prognostic biomarkers and demonstrate the heterogeneous expression pattern of CD200 in patients with pancreatic ductal adenocarcinoma. Abstract Pancreatic cancer is marked by a desmoplastic tumor microenvironment and low tumor immunogenicity, making it difficult for immunotherapy drugs to improve outcomes for patients. Tumor-infiltrating lymphocytes (TILs) and cancer-associated fibroblasts (CAFs) are seen in the tumor microenvironment of patients with pancreatic ductal adenocarcinoma (PDAC). In this work, we sought to characterize the expression levels and potential prognostic value of TILs (CD4, CD8, and CD20) and CAFs (Thy-1, FAP, and SMA) in a large retrospective cohort of PDAC patients. Additionally, we investigated the expression levels and prognostic significance of CD200, an immunoinhibitory protein that has shown interest as a potential target for immune checkpoint blockade. We measured the expression levels of these seven proteins with multiplexed immunofluorescence staining and quantitative immunofluorescence (QIF). We found CD8 and FAP to be independent predictors of progression-free survival and overall survival. CD200 was found to be heterogeneously expressed in both the tumor and stromal compartments of PDAC, with the majority of patients having positive stromal expression and negative tumor expression. This work demonstrates the potential clinical utility of CD8 and FAP in PDAC patients, and it sheds light on the expression patterns of CD200 in pancreatic cancer as the protein is being tested as a target for immune checkpoint blockade.

The objectives were to develop a standardized Ki-67 immunohistochemistry (IHC) method for precise, robust, and reproducible assessment of patients with early breast cancer, and utilize this assay to evaluate patients participating in the monarchE study (NCT03155997). The Ki-67 assay was developed and validated for sensitivity, specificity, repeatability, precision, and robustness using a predefined ≥20% cutoff. Reproducibility studies (intersite and intrasite, interobserver and intraobserver) were conducted at 3 external laboratories using detailed scoring instructions designed for monarchE. Using the assay, patient tumors were classified as displaying high (≥20%) or low (20%) Ki-67 expression; Kaplan-Meier methods evaluated 2-year invasive disease-free survival rates for these 2 groups among patients treated with endocrine therapy (ET) alone. All analytical validation and reproducibility studies achieved point estimates of90% for negative, positive, and overall percent agreement. Intersite reproducibility produced point estimate values of 94.7%, 100.0%, and 97.3%. External interobserver reproducibility produced point estimate values of 98.9%, 97.8%, and 98.3%. Among 1954 patients receiving ET alone, 986 (50.5%) had high and 968 (49.5%) had low Ki-67 expression. Patients with high Ki-67 had a clinically meaningful increased risk of developing invasive disease within 2 years compared with those with low Ki-67 [2-y invasive disease-free survival rate: 86.1% (95% confidence interval: 83.1%-88.7%) vs. 92.0% (95% confidence interval: 89.7%-93.9%), respectively]. This standardized Ki-67 methodology resulted in high concordance across multiple laboratories, and its use in the monarchE study prospectively demonstrated the prognostic value of Ki-67 IHC in HR+, HER2- early breast cancer with high-risk clinicopathologic features.

Highlights • PD-L1 expression was assessed for heterogeneity in 107 NSCLC patients. • Intra-tumoural heterogeneity was observed in 78% of cases. • Inter-tumoural heterogeneity was observed in 53% of cases. • 23% of cases had clinically relevant changes between primary and secondary tumours. • Sample site selection is an important consideration for testing PD-L1., Objectives PD-L1 expression on tumour cells can guide the use of anti-PD-1/PD-L1 immune modulators to treat patients with non-small cell lung cancer (NSCLC). Heterogeneity of PD-L1 expression both within and between tumour sites is a well-documented phenomenon that compromises its predictive power. Our aim was to better characterise the pattern and extent of PD-L1 heterogeneity with a view to optimising tumour sampling and improve its accuracy as a biomarker. Materials and methods Expression of PD-L1 was assessed by immunochemistry using the SP263 clone in 107 resected primary NSCLCs and their nodal metastases. Intra-tumoural heterogeneity, defined as ‘small-scale’ (mm²), ‘medium-scale’ (cm²) and ‘large-scale’ (between tumour blocks), was assessed by digital imaging using a novel ‘squares method’. Inter-tumoural heterogeneity between the primary tumours and their nodal metastases and between N1 and N2 nodal stages was also assessed. Results The majority of tumours demonstrated intra-tumoural heterogeneity (small-scale 78%, medium-scale 50%, large-scale 46%). Inter-tumoural heterogeneity between the primary and nodal metastases was present in 53% of cases and, in 17%, between N1 and N2 disease. These differences were occasionally sufficient to lead to discrepancy across the ≥1%, ≥25% and ≥50% cut-offs used to guide therapy. Conclusion Heterogeneity of PD-L1 expression is common, variable in scale and extent, and carries significant implications for its accuracy as a predictive biomarker. Extensive sampling reduces, but cannot eliminate, this inaccuracy.

CD200/CD200R is an immune checkpoint with broad expression patterns and a potential target for immune therapy. In this study, we assess both CD200 and CD200R expression in solid tumors, with a focus on lung cancer, and evaluate their association with clinicopathologic characteristics, mutation status, outcome, and programmed death-ligand 1 (PD-L1) expression. We used multiplexed quantitative immunofluorescence (QIF) to measure the expression of CD200 and CD200R in a total of 455 patients from three lung cancer cohorts. Using carefully validated antibodies, we performed target measurement with tyramide-based QIF panels and analyzed the data using the PM2000 microscope and AQUA software. CD200 tumor positivity was found in 29.7% of non-small cell lung cancer (NSCLC) patients and 33.3% of lung large cell neuroendocrine carcinoma (LCNEC) patients. CD200 demonstrated notable intratumoral heterogeneity. CD200R was expressed in immune cells in 25% of NSCLC and 41.3% of LCNEC patients. While CD200R is predominantly expressed in immune cells, rare tumor cell staining was seen in a highly heterogeneous pattern. CD200R expression in the stromal compartment was significantly higher in patients with squamous differentiation (p &lt, 0.0001). Neither CD200 nor CD200R were associated with other clinicopathologic characteristics or mutation status. Both biomarkers were not prognostic for disease-free or overall survival in NSCLC. CD200 showed moderate correlation with PD-L1. CD200/CD200R pathway is frequently expressed in lung cancer patients. Differential expression patterns of CD200 and CD200R with PD-L1 suggest a potential role for targeting this pathway alone in patients with NSCLC.

New therapeutics targeting immune checkpoint proteins have significantly advanced treatment of non-small cell lung cancer (NSCLC), but protein level quantitation of drug targets presents a critical problem. We used multiplexed, targeted mass spectrometry (MS) to quantify immunotherapy target proteins PD-1, PD-L1, PD-L2, IDO1, LAG3, TIM3, ICOSLG, VISTA, GITR, and CD40 in formalin-fixed, paraffin-embedded (FFPE) NSCLC specimens. Immunohistochemistry (IHC) and MS measurements for PD-L1 were weakly correlated, but IHC did not distinguish protein abundance differences detected by MS. PD-L2 abundance exceeded PD-L1 in over half the specimens and the drug target proteins all displayed different abundance patterns. mRNA correlated with protein abundance only for PD-1, PD-L1, and IDO1 and tumor mutation burden did not predict abundance of any protein targets. Global proteome analyses identified distinct proteotypes associated with high PD-L1-expressing and high IDO1-expressing NSCLC. MS quantification of multiple drug targets and tissue proteotypes can improve clinical evaluation of immunotherapies for NSCLC.

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.

Ectodomain shedding unleashes the aggressive nature of the MET oncogene product. Using specific C- and N-terminal MET antibodies (D1C2 and A2H2-3), MET protein status (i.e., no MET, decoy MET, transmembranous C-terminal MET with or without the ectodomain) was investigated in oral squamous cell carcinoma. For the cancers showing transmembranous C-terminal MET, the impact of ectodomain shedding on prognosis was investigated. To examine ectodomain shedding, reduced lysates of oral squamous cell carcinoma cell lines were immunoblotted using D1C2 and an ELISA was performed on culture media using A2H2-3. In addition, reduced lysates of fresh frozen tissues of 30 oral squamous cell carcinoma were immunoblotted using D1C2 and immunohistochemistry was performed on corresponding formalin-fixed paraffin-embedded tissues using both antibodies on parallel sections. To examine MET protein status, differences between membranous D1C2 and A2H2-3 immunoreactivities were scored using parallel tissue microarray sections representing 156 oral squamous cell carcinoma. The prognostic value of ectodomain shedding was examined using Cox regression analysis for disease-free survival and overall survival. Ectodomain shedding was observed in all cell lines, 43% (n = 13) of fresh frozen and 50% (n = 15) of formalin-fixed paraffin-embedded cancers (27% overlap, n = 8). The tissue microarray showed no MET in 23% (n = 36), decoy MET in 9% (n = 14), and transmembranous C-terminal MET in 68% (n = 106) of examined cancers. Within the latter group, ectodomain shedding occurs in 36% (n = 38) of the cases and is independently associated with poor disease-free survival (HR = 2.41; 95% CI, 1.35-4.30 and P = 0.003)-though not overall survival (HR = 1.64; 95% CI, 0.92-2.94 and P = 0.095)-after correcting for factors known to influence survival. In conclusion, MET ectodomain shedding occurs in transmembranous C-terminal MET positive oral squamous cell carcinoma and is independently associated with disease-free survival. These findings might aid in designing companion diagnostics for targeted therapies directed against MET.

Background: CD200 is a membrane-bound glycoprotein expressed on normal tissue, tumor and immune cells. The interaction of CD200 with its receptor leads to attenuation of the inflammatory process, resulting in tumor cell mediated immune suppression. CD200 has been characterized in hematologic malignancies and consists of an independent negative prognostic factor for chronic lymphocytic leukemia, acute myeloid leukemia and multiple myeloma patients. Here we assess both CD200 and CD200R expression in lung cancer and evaluate its association with clinicopathologic characteristics, mutation status, outcome and PD-L1 expression. Methods: We used quantitative multiplexed immunofluorescence (QIF) to measure the expression of CD200 and CD200R in 287 non-small cell lung cancer (NSCLC) patients and 30 patients with large cell neuroendocrine carcinomas (LCNEC) of the lung. We performed target measurement with tyramide-based QIF panels and analyzed the data using the PM2000 microscope and AQUA software. Targets were measured in cytokeratin-positive (CK+) tumor compartment and cytokeratin-negative (CK-) stromal compartment. Results: CD200 tumor positivity was found in 29.7% of NSCLC patients and 33.3% of LCNEC patients. CD200 demonstrated notable intratumoral heterogeneity (R2=0.09-0.31 between different TMA blocks versus 0.46-0.60 for CD200R). CD200R was expressed in immune cells in 25% of NSCLC and 41.3% of LCNEC patients. While CD200R is predominantly expressed in immune cells, rare tumor cell staining was seen in a highly heterogeneous pattern. CD200R expression in the stromal compartment was significantly higher in patients with squamous differentiation (p Conclusions: CD200 and CD200R pathway represents a novel immune checkpoint, frequently expressed in lung cancer patients. Co-expression patterns of CD200 and CD200R with PD-L1 suggest a potential role for targeting this pathway alone or in combination with PD-1/PD-L1 axis inhibitors. Citation Format: Ioannis A. Vathiotis, Tyler MacNeil, Jon Zugazagoitia, Sandra Martinez-Morilla, Fahad Ahmed, Konstantinos N. Syrigos, Aaron M. Gruver, Kyla Driscoll, David L. Rimm. Quantitative assessment of CD200 and CD200R expression in lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 937.

Background and objectives Although MET amplification/overexpression was observed in a subset of gastric cancer (GC) patients, the relationship between MET amplification/overexpression in primary GC and liver metastasis was unclear. Methods GC samples and matched liver metastases (N = 47) were analyzed by fluorescence/silver in-situ hybridization (FISH/SISH) and by immunohistochemistry for MET amplification and MET expression, respectively. MET-copy number (CN) and Met expression data from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD, N = 356) were also analyzed. Results Significant overlap existed between MET amplification and Met expression in both primary stomach tumors (P = 0.013) and liver metastasis (P = 0.001). In TCGA-STAD, MET-CN (≥4 copies) and MET expression were also positively correlated (r = 0.761; P = 0.017). Comparative analysis revealed a strong association between MET expression and MET amplification (85% concurrence) in primary stomach tumors and matched liver metastasis. MET status in synchronous liver metastasis (N = 36) was correlated with primary stomach tumors. However, a significant correlation between primary tumors and liver metastases was not observed in patients with metachronous liver metastasis. Survival analyses revealed that both MET amplification and MET overexpression were prognostic of poor outcomes. Conclusions MET amplification and Met overexpression were positively correlated in GC. MET status should be re-evaluated in GC patients with liver metastasis, especially for metachronous metastasis.

Objectives: To assess the feasibility of using a novel ultrasensitive bright-field in situ hybridization approach (BRISH) to evaluate κ and λ immunoglobulin messenger RNA (mRNA) expression in situ in B-cell non-Hodgkin lymphoma (NHL). Methods: A series of 110 semiconsecutive clinical cases evaluated for lymphoma with historic flow cytometric (FCM) results were assessed with BRISH. Results: BRISH light chain restriction (LCR) results were concordant with FCM in 108 (99%) of 109 evaluable cases. Additional small B-cell lymphoma cohorts were successfully evaluated. Conclusions: BRISH analysis of κ and λ immunoglobulin mRNA expression is a sensitive tool for establishing LCR in B-cell NHL when FCM results are not available.

// Bryce P Portier 1 , Eugen C Minca 2 , Zhen Wang 2 , Christopher Lanigan 2 , Aaron M Gruver 2 , Erinn Downs-Kelly 2 , G Thomas Budd 3 , and Raymond R Tubbs 2 1 Department of Pathology and Genomic Medicine, Houston Methodist, Houston, TX 2 Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH 3 Department of Solid Tumor Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH Correspondence: Bryce P. Portier, email: // Keywords : Breast carcinoma, HER2, HER4, Immunohistochemistry, RT-qPCR, Trastuzumab, H-Score Received : July 31, 2013 Accepted : August 24, 2013 Published : August 26, 2013 Abstract Prognostic and predictive markers utilized in invasive breast carcinoma are limited and include ER, PR, Ki67, and ERBB2 (HER2). In the case of HER2, over-expression or amplification serves as eligibility for anti-HER2 based therapy, including trastuzumab (Herceptin®, Genentech). While clinical trials have shown trastuzumab improves overall survival and time to progression, an individual’s response to anti-HER2 based therapy is highly variable. This suggests that, in a “uniform” HER2 positive population, additional markers could help in predicting patient outcome to therapy. Here we utilized a recently validated high-specificity HER4 antibody (E200) and generated a standard clinical HER4 scoring algorithm (HER4 H-Score) utilizing two breast carcinoma cohorts: 1) patients receiving neoadjuvant trastuzumab (n=47) and 2) patients receiving trastuzumab for metastatic disease (n=33). Our HER4 H-Score showed significant correlation with high sensitivity RT-qPCR performed on matched patients (p=

Acute promyelocytic leukemia (APL) is typically defined at the molecular level by a reciprocal translocation of the promyelocytic leukemia (PML) and retinoic acid receptor α (RARA) genes. An accurate diagnosis of APL is critical for appropriate choice of therapy and prognostic assessment. Cryptic and variant rearrangements in APL are discoverable by a variety of molecular methods including fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction, or gene sequencing. Rare reports of FISH-negative APL harboring cryptic rearrangements of PML-RARA detected by reverse transcriptase polymerase chain reaction or sequencing have been described. Here, we describe the detection of cryptic or variant PML-RARA rearrangements by translocation-based comparative genomic hybridization (tCGH), a recently described modification of traditional CGH technology that facilitates the detection of balanced translocations by means of the linear amplification of a potential translocation breakpoint region(s), in 2 unusual cases of APL. One tumor lacked detectable t(15;17) by karyotype and FISH, and the other tumor lacked the typical morphologic and immunophenotypic features of APL and had a variant 3-way translocation involving PML and RARA. PML-RARA translocations were identified by tCGH in both cases providing confirmation of the diagnosis of APL. These data emphasize the benefit of using complementary molecular methods including tCGH for detecting cryptic and variant PML-RARA translocations in unusual cases of APL.

Context.—Distinction between primary lung carcinomas and metastases from other sites, especially the urinary tract, is a common diagnostic dilemma. As urothelial carcinomas can demonstrate a broad range of morphology and frequently demonstrate squamous differentiation, discerning metastatic urothelial carcinoma to the lung from primary pulmonary squamous cell carcinoma can be challenging. Objective.—To investigate immunostains that may aid in the distinction of urothelial carcinoma metastatic to the lung. Design.—Staining patterns of 14 markers in primary urothelial carcinoma of the bladder and primary squamous cell carcinoma of the lung were examined to establish a diagnostic panel. These antibodies were subsequently tested on tumors taken from 30 patients with a paired urinary tract and metastatic lung lesion. Results.—The best markers to distinguish poorly differentiated metastatic urothelial carcinoma from primary pulmonary squamous cell carcinoma were CK7, CK20, GATA-3, CK14, desmoglein-3, and uroplakin III, with the utility of the latter dependent upon the quantity of tissue available for analysis. The observed percentage positive staining in nonmetastatic urothelial carcinoma versus primary pulmonary squamous cell carcinoma with these antibodies was as follows: CK7 (100% versus 33%), CK20 (54% versus 7%), GATA-3 (78% versus 23%), CK14 (32% versus 77%), desmoglein-3 (11% versus 87%), and uroplakin III (14% versus 0%). Similar expression patterns were observed among the paired cases. Conclusion.—When interpreted in correlation with clinical history and histomorphology, a panel of immunostains including CK7, CK20, GATA-3, CK14, desmoglein-3, and uroplakin III may be a useful adjunct in the distinction of metastatic urothelial carcinoma to the lung.

Cardiac lymphomas are rare, and the spectrum of pathologic features is not well defined. We encountered an unusual case of cardiac lymphoma residing within a presumed thrombus. To place such cases in context, we reviewed all cardiac lymphomas presenting to a large US cardiovascular medicine referral center during a 30-year period. A total of 14 cardiac lymphomas were identified, and these included 6 primary cardiac lymphomas (PCLs) and 8 lymphomas secondarily involving cardiac structures. Upon review, 3 of the PCLs were confirmed to be diffuse large B-cell lymphoma, not otherwise specified, involving the myocardium. The other 3 cases of PCL lacked myocardial invasion and showed lymphoma cells embedded in fibrin thrombus. Acute inflammation was not evident. These lymphomas presented in immunocompetent male individuals and involved either a prolapsed myxomatous mitral valve, a pseudomyxoma from the left atrium, or a thrombus arising in a synthetic aortic root graft. All 3 consisted of large atypical lymphocytes expressing a nongerminal center B-cell immunophenotype. Two cases were positive for Epstein-Barr virus (latency type III), but none demonstrated human herpes virus-8 latent nuclear antigen. No systemic disease was found at presentation or during follow-up. In our experience, fibrin-associated large B-cell lymphoma arising in the heart represents a substantial proportion of PCL. These lymphomas appear to represent an underrecognized variant of diffuse large B-cell lymphoma with favorable outcome. Further study is needed to understand their natural history.

Context.—Adenocarcinoma of the breast is the most frequent cancer affecting women in both developed and developing regions of the world. From the moment of clinical presentation until the time of pathologic diagnosis, patients affected by this disease will face daunting questions related to prognosis and treatment options. While improvements in targeted therapies have led to increased patient survival, these same advances have created the imperative to accurately stratify patients to achieve maximum therapeutic efficacy while minimizing side effects. In this evolving era of personalized medicine, there is an ever-increasing need to overcome the limitations of traditional diagnostic practice.Objective.—To summarize the molecular diagnostics traditionally used to guide prognostication and treatment of breast carcinomas, to highlight published data on the molecular classification of these tumors, and to showcase molecular assays that will supplement traditional methods of categorizing the disease.Data Sources.—A review of the literature covering the molecular diagnostics of breast carcinomas with a focus on the gene expression and array studies used to characterize the molecular signatures of the disease. Special emphasis is placed on summarizing evolving technologies useful in the diagnosis and characterization of breast carcinoma.Conclusions.—Available and emerging molecular resources will allow pathologists to provide superior diagnostic, prognostic, and predictive information about individual breast carcinomas. These advances should translate into earlier identification and tailored therapy and should ultimately improve outcome for patients affected by this disease.

Autosomal dominant retinocerebral vasculopathy with cerebral leukodystrophy (RVCL) is a rare neurovascular syndrome causing retinal and central nervous system vasculopathy often recognized as contrast-enhancing white matter changes or pseudotumors on imaging. Heterozygous frameshift mutations in the 3-prime repair exonuclease 1 gene have been identified in families affected by RVCL. Variable light microscopic findings and a characteristic ultrastructural appearance of the vasculature in the brain have been reported. Description of the ophthalmic histopathology is exceedingly rare. Here, we report previously undescribed bilateral eye findings in a patient diagnosed with RVCL. The ophthalmic pathology includes thickening and reduplication of the retinal capillary basal lamina demonstrated by electron microscopy. These findings expand what is known about this disease and help further delineate its phenotype.

Assessment of ERBB2 (HER2) status in breast carcinomas has become critical in determining response to the humanised monoclonal antibody trastuzumab. The current joint College of American Pathologists and the American Society of Clinical Oncology guidelines for the evaluation of HER2 status in breast carcinoma involve testing by immunohistochemistry and fluorescence in situ hybridisation (FISH). However, neither of these modalities is without limitations. Novel bright field in situ hybridisation techniques continue to provide viable alternatives to FISH testing. While these techniques are not limited to evaluation of the HER2 gene, the extensive number of studies comparing bright field in situ techniques with other methods of assessing HER2 status allow a robust evaluation of this approach. Analysis of the literature demonstrates that, when used to assess HER2 gene status, bright field in situ hybridisation demonstrates excellent concordance with FISH results. The average percentage agreement in an informal analysis of studies comparing HER2 amplification by chromogenic in situ hybridisation with FISH was 96% (SD 4%); kappa coefficients ranged from 0.76 to 1.0. Although a much smaller number of studies are available for review, similar levels of concordance have been reported in studies comparing HER2 amplification by methods employing metallography (silver in situ hybridisation) with FISH. A summary of the advancements in bright field in situ hybridisation, with focus on those techniques with clinical applications of interest to the practicing pathologist, is presented.

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co-precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1-D SDS-PAGE of co-precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co-precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.

Background: Breast carcinomas that demonstrate a heterogeneous ERBB2 (HER2) status or equivocal results by both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) present diagnostic challenges for which there is neither a standard methodology to achieve resolution in the clinical laboratory nor a uniform approach to management. We assessed the feasibility of using a novel automated and quantitative HER2 mRNA bright field in situ hybridization (ISH) assay capable of single molecule detection to determine HER2 status in invasive breast carcinomas that demonstrated significant tumor heterogeneity or failed to be resolved by standard IHC and FISH algorithmic testing. Design: Formalin-fixed, paraffin-embedded (FFPE) breast carcinomas from a non-consecutive series of 163 patients were analyzed for HER2 mRNA using a fully automated bright field RNA ISH assay (RNAscope, Advanced Cell Diagnostics, Hayward, CA). Cases were assigned into either a training set (n = 34) or a validation set (n = 129) and analyzed by both Q-RT-PCR and RNAscope. automated image analysis was used to numerate the punctate signal dots per cell in RNAscope-stained slides. A HER2 mRNA score based on single-cell quantification by RNAscope was developed and correlated to HER2 FISH and HER2 mRNA Q-RT-PCR results. A simple cutoff value was derived using the training set and applied to the validation set. Results: Evaluable HER2 results were obtained for 154 cases (94.5%) by RNAscope and 163 cases (100%) by Q-RT-PCR. In the training set, both FISH/IHC positive and negative cases were definitively separated by both Q-RT-PCR and RNAscope. HER2 mRNA dots per cell correlated strongly to FISH (Spearman r=0.77) and Q-RT-PCR (r = 0.81). Application of both methods to the validation set resulted in correct identification of 31/31 positive cases and 41/43 negative (overall concordance=97.3%) for both RNAscope and Q-RT-PCR. RNAscope showed a significant advantage over Q-RT-PCR in correctly identifying cases equivocal by FISH that were resolved by reflex IHC testing. RNAscope classified 7 of 26 (26.9%) FISH/IHC double equivocal cases as positives. In cases with HER2 protein heterogeneity, RNAscope showed a 100% concordance with FISH results, whereas Q-RT-PCR showed a 42.9% concordance. Conclusion: RNAscope analysis of HER2 mRNA is an effective means to resolve HER2 status in double equivocal cases and cases that demonstrate heterogeneity. Automation and image analysis-based quantification minimize analytical and post-analytical variability. Quantification of single RNA transcripts in situ at single-cell level demonstrates superiority over qRTPCR and great potential in predictive biomarker analysis. Further studies of larger cohorts correlating clinical response with HER2 mRNA expression in situ are warranted. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD02-04.

9019 Background: MET expression is a mechanism of resistance to EGFR inhibition in EGFRmt NSCLC and correlated with poor prognosis. Emi (LY2875358) is a humanized IgG4 monoclonal bivalent MET antibody that blocks ligand dependent and independent HGF/MET signaling. This Phase 2 study compared the clinical activity of Emi + E versus single agent E in 1st line EGFRmt metastatic NSCLC. Methods: Stage IV, EGFRmt NSCLC pts with disease control following an 8-week lead-in E (150 mg PO QD) treatment were randomized 1:1 to receive Emi (750 mg IV Q2W) + E or E alone. Pts were stratified by ECOG PS, ethnicity, MET expression status, and response at the end of the lead-in. The primary endpoint was PFS from randomization. Additional endpoints included safety, OS, PK, and exploratory analysis of MET-expressing populations. Results: Out of181 pts enrolled, 141 pts were randomized (Emi+E: 71; E: 70). In the ITT population, median PFS for EMI+E was 9.3 months (m) compared with 9.5 m for E (HR = 0.89: 90% CI 0.64-1.23; p = 0.534). Exploratory analysis of MET-high expressing pts (MET 3+ expression in ≥90% of tumor cells; n = 24 pts) showed a 15.3 m improvement in PFS (EMI+E: 20.7 m; E: 5.4 m [HR: 0.39; 90% CI: 0.17-0.91]). No difference in PFS was observed in the complementary population (HR: 1.1 [90% CI: 0.7-1.7]). Similar frequencies of related AEs were reported for both treatment arms. Drug-related TEAEs that were more frequent ( > 10%) for Emi+E were peripheral edema and fatigue (all grade 1 or 2). Emi serum concentrations were consistent with previously obtained PK results, and no apparent exposure-response was observed. Median OS in the ITT population was not achieved (NA) for either arm. In MET-high expressing pts, median OS was 20.6 m for E (90% CI: 8.87, NA) whereas it was not achieved for Emi+E (90% CI: NA, NA). Conclusions: No statistically significant difference in PFS was noted in the ITT population.Exploratory analysis confirmed that high MET expression is a negative prognostic marker for pts treated with E and indicated that these pts may receive clinically meaningful benefit from Emi+E. Clinical trial information: NCT01897480.

Development of novel targeted therapies directed against hepatocyte growth factor (HGF) or its receptor (MET) necessitates the availability of quality diagnostics to facilitate their safe and effective use. Limitations of some commercially available anti-MET antibodies have prompted development of the highly sensitive and specific clone A2H2-3. Here we report its analytical properties when applied by an automated immunohistochemistry method.Excellent antibody specificity was demonstrated by immunoblot, ELISA, and IHC evaluation of characterised cell lines including NIH3T3 overexpressing the related kinase MST1R (RON). Sensitivity was confirmed by measurements of MET in cell lines or characterised tissues. IHC correlated well with FISH and quantitative RT-PCR assessments of MET (P 0.001). Good total agreement (89%) was observed with the anti-MET antibody clone SP44 using whole-tissue sections, but poor positive agreement (21-47%) was seen in tissue microarray cores. Multiple lots displayed appropriate reproducibility (R(2) 0.9). Prevalence of MET positivity by IHC was higher in non-squamous cell NSCLC, MET or EGFR amplified cases, and in tumours harbouring abnormalities in EGFR exon 19 or 21.The anti-MET antibody clone A2H2-3 displays excellent specificity and sensitivity. These properties make it suitable for clinical trial investigations and development as a potential companion diagnostic.

Diagnostics in the field of breast carcinoma are constantly evolving. The recent wave of molecular methodologies, both microscope and non-microscope based, have opened new ways to gain insight into this disease process and have moved clinical diagnostics closer to a ‘personalized medicine’ approach. In this review we highlight some of the advancements that laboratory medicine technology is making toward guiding the diagnosis, prognosis, and therapy selection for patients affected by breast carcinoma. The content of the article is largely structured by methodology, with a distinct emphasis on both microscope based and non-microscope based diagnostic formats. Where possible, we have attempted to emphasize the potential benefits as well as limitations to each of these technologies. Successful molecular diagnostics, applied in concert within the morphologic context of a patient's tumor, are what will lay the foundation for personalized therapy and allow a more sophisticated approach to clinical trial stratification. The future of breast cancer diagnostics looks challenging, but it is also a field of great opportunity. Never before have there been such a plethora of new tools available for disease investigation or candidate therapy selection.

Homologous recombination (HR) is a mechanism for repairing DNA interstrand crosslinks and double-strand breaks. In mammals, HR requires the activities of the RAD51 family (RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1), each of which contains conserved ATP binding sequences (Walker Motifs A and B). RAD51D is a DNA-stimulated ATPase that interacts directly with RAD51C and XRCC2. To test the hypothesis that ATP binding and hydrolysis by RAD51D are required for the repair of interstrand crosslinks, site-directed mutations in Walker Motif A were generated, and complementation studies were performed in Rad51d-deficient mouse embryonic fibroblasts. The K113R and K113A mutants demonstrated a respective 96 and 83% decrease in repair capacity relative to wild-type. Further examination of these mutants, by yeast two-hybrid analyses, revealed an 8-fold reduction in the ability to associate with RAD51C whereas interaction with XRCC2 was retained at a level similar to the S111T control. These cell-based studies are the first evidence that ATP binding and hydrolysis by RAD51D are required for efficient HR repair of DNA interstrand crosslinks.

Homologous recombination is a double-strand break repair pathway required for resistance to DNA damage and maintaining genomic integrity. In mitotically dividing vertebrate cells, the primary proteins involved in homologous recombination repair are RAD51 and the five RAD51 paralogs, RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. In the absence of Rad51d, human and mouse cells fail to proliferate, and mice defective for Rad51d die before birth, likely as a result of genomic instability and p53 activation. Here, we report that a p53 deletion is sufficient to extend the life span of Rad51d-deficient embryos by up to 6 days and rescue the cell lethal phenotype. The Rad51d−/− Trp53−/− mouse embryo–derived fibroblasts were sensitive to DNA-damaging agents, particularly interstrand cross-links, and exhibited extensive chromosome instability including aneuploidy, chromosome fragments, deletions, and complex rearrangements. Additionally, loss of Rad51d resulted in increased centrosome fragmentation and reduced levels of radiation-induced RAD51-focus formation. Spontaneous frequencies of sister chromatid exchange were not affected by the absence of Rad51d, but sister chromatid exchange frequencies did fail to be induced upon challenge with the DNA cross-linking agent mitomycin C. These findings support a crucial role for mammalian RAD51D in normal development, recombination, and maintaining mammalian genome stability.

Cholangiocarcinoma (CCA) originates from biliary tract epithelium and can be classified anatomically into intrahepatic or extrahepatic CCA. Although a relatively rare disease in the United States and Europe, the incidence of intrahepatic CCA is on the rise with unknown causes. CCA incidence is higher in Asia and the etiology is associated with infections such as liver fluke and hepatitis B/C. Prognosis at diagnosis is poor with median survival time of < 1 year, and only 10-20% of patients are eligible for tumor resection at diagnosis. This study examined the prevalence of MET over-expression, activating single point mutations of KRAS and IDH1/2, and ROS1 gene fusions in ∼100 intrahepatic and extrahepatic CCA FFPE tumor tissues obtained from non-Asian (n=40) and Asian (n=60) patients. Immunohistochemistry performed with an anti-MET-specific antibody (A2) demonstrated that MET is expressed in the majority of all intrahepatic CCA samples analyzed, with ∼50% of samples reported with membranocytoplasmic scores of 2+ (moderate intensity) or 3+ (strong intensity) on a 0-3+ scale. Interestingly, most non-malignant bile ducts and much vascular endothelia also stained lightly positive for MET. To determine mutation frequencies of KRAS and IDH1/2, competitive allele-specific Taqman® PCR (castPCR) was performed on DNA extracted from FFPE CCA tissue (limit of mutation detection 0.1%). Fourteen KRAS activating mutations (G12S, G12R, G12A, G13C, G13S, G13R, G12D, G13D, G12V, G12C, Q61R, Q61L, Q61H (c.183A>C and c.183A>T for Q61H), 5 IDH1 mutations (R132G, R132S, R132H, R132C, R132L), and 5 IDH2 mutations (R172G, R172M, R172K, R172W, R172S) were analyzed. Overall, 25% of analyzed samples were positive for KRAS mutation, and G12D was the predominant mutation (∼60%). One-third of Asian samples were positive for KRAS mutation, whereas less than one-fifth of non-Asian samples contained KRAS mutations. For IDH1, the frequency of mutation was less than 10% overall, and the majority of patients with IDH1 mutations were non-Asian. The R132C mutation was the predominant IDH1 mutation, and all tissues that were positive for IDH1 mutations were of intrahepatic origin. Interestingly, 2 out of the 7 samples positive for IDH1 mutations (R132C) were also positive for G12D KRAS mutation. There is no trend of MET expression correlating with either KRAS or IDH1 mutations. IDH2 analyses by castPCR and FISH studies examining ROS1 gene fusion are ongoing. Based on these data, inhibitors of receptor tyrosine kinases and their signaling pathways such as MET and ROS1 may merit clinical evaluation in CCA patients. LY2801653, a MET inhibitor which also has inhibitory activity against ROS1 and MKNK1/2 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037). Citation Format: Megan N. Thobe, S. Betty Yan, Kelly M. Credille, Aejaz Nasir, Jessica A. Roseberry Baker, Mary Lajiness, Nathan A. Brooks, Darryl W. Ballard, Donna M. Farley, Victoria L. Peek, Suzane L. Um, G. Jason Jin, Raymond Gilmour, Christoph Reinhard, Jeremy R. Graff, Andrew E. Schade, Aaron M. Gruver, Bruce Colligan, Larry Douglass, Julia Carter, Richard A. Walgren. Prevalence of MET expression, activating mutations of KRAS and IDH1/2, and ROS1 fusions in cholangiocarcinoma patient tumor samples. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2339. doi:10.1158/1538-7445.AM2013-2339

4263 Background: COMPLETE is a registry of peripheral T-cell lymphoma (PTCL) patients from academic and community settings in the US. For the registry, selected biomarkers are recorded to determine which markers may have informed the diagnosis of PTCL. Research staff at participating sites enter data from pathology reports into an electronic data capture (EDC) system; however, pathology reports can be difficult to interpret, particularly for those who have not received specialized training. To ensure that biomarker data in COMPLETE accurately reflects pathology reports, a biomarker QA initiative was undertaken. A similar initiative was pursued by the International T-Cell Project and is the subject of a separate, parallel abstract. Methods: The COMPLETE biomarker QA initiative includes review of on-site data entered for 26 biomarkers commonly assessed in the diagnostic work-up of PTCL. External review by a board-certified pathologist is triggered once a site has entered the biomarker data into the EDC system for enrolled patients and uploaded the de-identified pathology reports. All patients enrolled at a site within 30 days of the first patient were reviewed. A single mismatch between the site-entered data and the reviewer's findings counts as an error. Findings of the review are entered into the EDC system to facilitate analysis and queries are sent to the site in cases where corrections are needed. Results: Data for 119 patients from 43 centers comprising a sum of 3570 biomarker entries were reviewed by an external hematopathologist. The mean number of phenotypic markers assessed per patient was 12 (range: 4–20), and the mean number of gene rearrangement tests performed was 0.2 (range: 0–3). Of the 119 patients, 13 (11%) had no EDC entry errors, 33 (28%) had 1–2 errors, 34 (29%) had 3–5 errors, 31 (26%) had 6–10 errors and the remaining 8 cases (7%) had 11–15 errors. The reviewer agreed with 74% of entries where the site noted a finding was positive; of the remaining entries where the site indicated the findings were positive, the reviewer noted they were negative (12%), not assessed (10%) or indeterminate (5%). For entries where the site indicated the finding was negative, the reviewer was in agreement 79% of the time; for the remaining 21% of negative-reported entries by the site, the reviewer indicated the marker was not assessed (12%), indeterminate (6%) or positive (3%). The reviewer was in agreement with the site in 92% of the entries where the site indicated the marker was not assessed; otherwise, the reviewer found the marker was negative (4%), positive (3%) or indeterminate (1%). The markers accounting for the most errors (each marker accounting for 5% or more of errors) were CD8, CD20, CD30, Epstein-Bar Virus (EBV) and T-cell receptor gamma gene rearrangement (TCR-g). The types of errors for these markers are noted in the table below. | Marker, N (%) Errors Overall | Type of Error Noted by the Reviewer for Markers with the Most Errors, n (%) | |:-------------------------------------------:| --------------------------------------------------------------------------- | ---------------------------------------------- | | Finding is recorded in non-neoplastic cells | Pathology report unclear | Test performed, result not in pathology report | Marker is positive or negative; site notes opposite | Marker is not assessed or indeterminate; site notes positive or negative result | Marker is positive or negative; site notes marker was not assessed | Other | | CD8, 40 (8%) | 1 (3%) | 12 (30%) | 2 (5%) | 11 (28%) | 7 (18%) | 6 (15%) | 1 (3%) | | CD20, 47 (9%) | 24 (51%) | 6 (13%) | 0 (0%) | 5 (11%) | 6 (13%) | 6 (13%) | 0 (0%) | | CD30, 25 (5%) | 5 (20%) | 3 (12%) | 1 (4%) | 3 (12%) | 6 (24%) | 7 (28%) | 0 (0%) | | EBV, 27 (5%) | 1 (4%) | 2 (7%) | 0 (0%) | 0 (0%) | 0 (0%) | 23 (85%) | 1 (4%) | | TCR-g, 24 (5%) | 0 (0%) | 4 (17%) | 0 (0%) | 0 (0%) | 5 (21%) | 12 (50%) | 3 (13%) | Sum may exceed 100% due to rounding. Recording the type of EBV test performed [in situ hybridization (ISH) and immunohistochemistry (IHC)] presented further challenges, accounting for 8% of errors. Common errors included: 1) the site indicated the test was not performed when it was performed per report and 2) the site indicated a particular test (ISH, IHC) was performed but the method used was unclear in the report. Conclusions: Results of the COMPLETE biomarker QA analysis indicate that errors in interpretation or recording of biomarker data from pathology reports are common. For registries and other observational studies, periodic reviews by an external pathologist may guide site training and improve the accuracy of data collection. For interventional clinical trials in PTCL, central review of all biomarker data by expert hematopathologists should be considered. Disclosures: Hsi: Allos: Research Funding; Eli Lilly: Research Funding; Abbott: Research Funding; Cellerant Therapeutics: Research Funding; BD Biosciences: Research Funding; Millenium: Research Funding. Gruver: MedNet Solutions: Honoraria. Foss: Allos: Consultancy; Celgene: Consultancy; Seattle Genetics: Consultancy; Eisai: Consultancy; Celgene: Study Grant, Study Grant Other; Merck: Study Grant, Study Grant Other. Carson: Allos: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Pinter-Brown: Allos: Consultancy, Membership on an entity's Board of Directors or advisory committees. Horwitz: Allos: Consultancy, Research Funding. Rosen: Allos: Consultancy, Honoraria. Pro: Celgene: Honoraria, Research Funding; Spectrum : Honoraria; Allos: Honoraria; Seattle Genetics : Research Funding. Gisselbrecht: Allos: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Abstract 2529 The clinical heterogeneity of acute myeloid leukemia (AML) is well established and identification of specific balanced translocations is a critical component of the classification of AML. Patients with translocations are assigned to prognostic groups generally defined as good [e.g., t(8;21)(q22;q22), inv(16) (p13q22), t(15;17)(q24;q21)], intermediate [e.g., t(3;5)(q25;q34), t(9;11)(p22;q23)] and poor [e.g., other MLL translocations, inv(3)(q21q26.2), t(6;9)(p23;q34)]. Further refinement based on the identification of new genetic markers may help tailor prognostic and treatment decisions. In an effort to identify new molecular markers associated with recurrent translocations, we have analyzed 30 cases of AML with apparently balanced translocations using a microarray-based technology coupled with linear DNA amplification [translocation-CGH (tCGH)], providing high resolution mapping of translocation breakpoints and identification of copy number abnormalities at those breakpoints (BkPt-CNAs). Analysis can be multiplexed to interrogate 14 balanced translocations in a single test. Additionally, all cases were assayed by high-density aCGH to identify additional clinically relevant copy number abnormalities (CNAs) throughout the genome. For each sample these test results were compared. Patients were all adults (>40 years of age), 10 males and 20 females, and included t(15;17)(q24;q21) (8 cases), t(8;21) (q22;q22) (6 cases), inv(16)(p13q22) (5 cases), t(9;11)(p22;q23) (5 cases), t(3;5)(q25;q34) (4 cases), t(6;11)(q27;q23) (1 case) and t(6;9)(p23;q34) (1 case). Of the 30 cases, 6 showed BkPt-CNAs in one or both translocation partners. All 6 (100%) of these cases exhibited additional clinically relevant CNAs, whereas only 11/24 (46%) cases with no BkPt-CNAs showed additional CNAs. Thus, BkPt-CNAs are significantly correlated with the presence of secondary clinically relevant alterations (p=0.0208). Importantly, no MLL translocations cases revealed BkPt-CNAs. In contrast, 3 of 4 t(3;5)(q25;q34) cases showed BkPt-CNAs in both genes (NPM1 and MLF1) and additional CNAs, with two cases showing large deletions (>0.5 Mb) at both breakpoints. Alterations involved RSRC1 (mRNA splicing), FBXW11 (E3 ubiquitin protein ligase), STK10 (serine/threonine kinase), FGFR3 (growth factor receptor) and MGMT (O-6-methylguanine-DNA methyltransferase), findings potentially relevant to disease progression and treatment response. Cases of t(15;17), t(8;21) and inv(16) each showed a single case with BkPt-CNAs and these correlated with greater complexity for additional CNAs. For t(15;17), that case showed partial duplication of both the PML and RARA genes (47 kb and 39 kb) yielding a cryptic fusion (missed by karyotype and FISH) that produced the “long” form (bcr1) transcript based on the PML breakpoint and confirmed by rtPCR. This case also had a 24.56 Mb deletion at 13q14.2->q21.33 involving RB1, MIR15A and MIR16-1. In the 7 remaining PML-RARA cases [4 short (bcr3) and 3 long (bcr1)] with no BkPt-CNAs, two cases showed non-complex CNAs [(e.g., -Y (bcr1) or +8 (bcr3)]. Five of six t(8;21) cases exhibited CNAs, but the case with a BkPt-CNA (39 kb within RUNX1) exhibited greater complexity for CNAs (multiple gains and losses over >70 MB of distal 9q) than other cases (e.g., +8 or +15). Similarly, analysis of inv(16) cases demonstrated CNAs in 3 of 5 cases, with the greatest complexity in the case with BkPt-CNAs [deletion in CBFB and MYH11 involving ABCC1 (anion transporter implicated in multiple drug resistance), deletions 9q34.13 involving ABL1 and NUP214], while other cases had single changes of a homozygous deletion of GSTT1 or a heterozygous deletion of the 16p11.2 microdeletion syndrome region, either potentially inherited. The sole case of t(6;9) had only the relevant translocation. In conclusion, analysis of AML cases with balanced translocation by t-CGH and aCGH revealed significant correlation between BkPt-CNAs and the presence of secondary CNAs. Moreover, when BkPt-CNAs were present, the molecular complexity of additional CNAs was increased. This observation held true across various translocations evaluated, with the notable exception of MLL translocations. Thus BkPt-CNAs provide a broadly applicable signature for a subtle molecular instability in AML that, acquired or inherited, may predate translocations and may prove relevant to treatment response and outcome. Disclosures: Schultz: Signature Genomic Laboratories: Employment. Campbell:Abbott Molecular: Honoraria. Furrow:Signature Genomic Laboratories: Employment. Shaffer:Signature Genomic Laboratories: Employment. Ballif:Signature Genomic Laboratories: Employment.

Background The homologous recombination (HR) pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are essential for this process in vertebrates, and the RAD51D paralog is unique in that it participates in both HR repair and telomere maintenance. RAD51D is also known to directly interact with the RAD51C and XRCC2 proteins. Rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in HR regulation. The present study evaluated the interaction of the Rad51d splice isoform products with RAD51C and XRCC2 and their expression patterns. Results Yeast-2-hybrid analysis was used to determine that the Mus musculus Rad51d splice variant product RAD51DΔ7b (deleted for residues 219 through 223) was capable of interacting with both RAD51C and XRCC2 and that RAD51D+int3 interacted with XRCC2. In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2. Cellular localization, detected by EGFP fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. However, none of the splice variants were capable of restoring resistance of Rad51d-deficient cell lines to mitomycin C. RT-PCR expression analysis revealed that Rad51dΔ3 (deleted for exon 3) and Rad51dΔ5 (deleted for exon 5)transcripts display tissue specific expression patterns with Rad51dΔ3 being detected in each tissue except ovary and Rad51dΔ5 not detected in mammary gland and testis. These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10. Conclusion These results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.