Targeted Quantitative Mass Spectrometry Analysis of Protein Biomarkers From Previously Stained Single Formalin-Fixed Paraffin-Embedded Tissue Sections.

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-µm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine-based immunohistochemical staining. We analyzed serial unstained and stained sections from non-small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted ß-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points.

Population-based estimate for the correlation of the Oncotype Dx Breast Recurrence Score® result and Ki-67 IHC MIB-1 pharmDx in HR+, HER2-, node-positive early breast cancer.

BACKGROUND: The United States Food and Drug Administration recently approved a Ki-67 immunohistochemistry (IHC) assay to identify patients with early breast cancer at high disease recurrence risk. The Oncotype Dx Breast Recurrence Score® assay has been validated in hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) invasive breast cancer (IBC) to predict chemotherapy benefit and distant recurrence risk, regardless of nodal status. This study assessed the correlation between Recurrence Score® (RS) results and the Ki-67 IHC MIB-1 pharmDx assay. METHODS: HR+, HER2-, N1 IBC samples with RS results were examined by Ki-67 IHC; 311 specimens were collected, including 275 without regard to RS ("unselected RS") and 36 more with RS 26-100; 12 were lymph node negative upon pathology report review, and one had no Ki-67 score, leaving 262 unselected RS and 298 total samples. Spearman rank correlation was calculated using the unselected samples and a weighted rank correlation using all samples. A receiver operating characteristic (ROC) curve for predicting high RS (26-100) from Ki-67 was constructed. RESULTS: The Spearman rank correlation between Ki-67 and RS results was moderately positive (unselected RS samples: 0.396; 95% confidence interval [CI] 0.288-0.493; all samples: 0.394; 95% CI 0.294-0.486). While 71% of samples with RS 26-100 had Ki-67 ≥ 20%, 75% with RS 0-25 had Ki-67 < 20%. ROC area under the curve was 0.792 (95% CI 0.725-0.859). CONCLUSIONS: The moderately positive correlation is consistent with previous analyses suggesting the Oncotype Dx® assay and Ki-67 IHC MIB-1 assay should not be used interchangeably in clinical practice.

Accelerated instability testing reveals quantitative mass spectrometry overcomes specimen storage limitations associated with PD-L1 immunohistochemistry.

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 < 0.001), with MS demonstrating no loss of PD-L1 protein, even in sections with significant signal loss by IHC impacting diagnostic category. Clones 22C3 and 28-8 were most susceptible to signal loss, with E1L3N demonstrating the most robust signal (56%, 58%, and 33% reduction respectively; p < 0.05). Increased humidity and temperature resulted in significant acceleration of immunoreactivity loss, which was mitigated by storage with desiccant. MS demonstrated only modest oxidation of 274 methionine-containing peptides and aligned with IHC results suggesting peptide oxidation is not a major factor. These data imply immunoreactivity loss driven by humidity and temperature results in structural distortion of epitopes rendering them unsuitable for antibody binding following epitope retrieval. Limitations of IHC biomarker analysis from stored tissue sections may be mitigated by cost-effective use of desiccant when appropriate. In some scenarios, complementary MS is a preferred approach for retrospective analyses of archival FFPE tissue collections.

MET ectodomain shedding is associated with poor disease-free survival of patients diagnosed with oral squamous cell carcinoma.

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.

MET in gastric cancer with liver metastasis: The relationship between MET amplification and Met overexpression in primary stomach tumors and liver metastasis.

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.

Immunohistochemical application of a highly sensitive and specific murine monoclonal antibody recognising the extracellular domain of the human hepatocyte growth factor receptor (MET).

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Pathologist-trained machine learning classifiers developed to quantitate celiac disease features differentiate endoscopic biopsies according to modified marsh score and dietary intervention response.

BACKGROUND: Histologic evaluation of the mucosal changes associated with celiac disease is important for establishing an accurate diagnosis and monitoring the impact of investigational therapies. While the Marsh-Oberhuber classification has been used to categorize the histologic findings into discrete stages (i.e., Type 0-3c), significant variability has been documented between observers using this ordinal scoring system. Therefore, we evaluated whether pathologist-trained machine learning classifiers can be developed to objectively quantitate the pathological changes of villus blunting, intraepithelial lymphocytosis, and crypt hyperplasia in small intestine endoscopic biopsies. METHODS: A convolutional neural network (CNN) was trained and combined with a secondary algorithm to quantitate intraepithelial lymphocytes (IEL) with 5 classes on CD3 immunohistochemistry whole slide images (WSI) and used to correlate feature outputs with ground truth modified Marsh scores in a total of 116 small intestine biopsies. RESULTS: Across all samples, median %CD3 counts (positive cells/enterocytes) from villous epithelium (VE) increased with higher Marsh scores (Type 0%CD3 VE = 13.4; Type 1-3%CD3 VE = 41.9, p < 0.0001). Indicators of villus blunting and crypt hyperplasia were also observed (Type 0-2 villous epithelium/lamina propria area ratio = 0.81; Type 3a-3c villous epithelium/lamina propria area ratio = 0.29, p < 0.0001), and Type 0-1 crypt/villous epithelial area ratio = 0.59; Type 2-3 crypt/villous epithelial area ratio = 1.64, p < 0.0001). Using these individual features, a combined feature machine learning score (MLS) was created to evaluate a set of 28 matched pre- and post-intervention biopsies captured before and after dietary gluten restriction. The disposition of the continuous MLS paired biopsy result aligned with the Marsh score in 96.4% (27/28) of the cohort. CONCLUSIONS: Machine learning classifiers can be developed to objectively quantify histologic features and capture additional data not achievable with manual scoring. Such approaches should be further investigated to improve biopsy evaluation, especially for clinical trials.

Two Instrument Comparison of Reagents From a US FDA-Approved Assay for the Assessment of Ki-67 in High-Risk Early Breast Cancer.

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 average <1% 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 RS >25. 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.

A Standardized Investigational Ki-67 Immunohistochemistry Assay Used to Assess High-Risk Early Breast Cancer Patients in the monarchE Phase 3 Clinical Study Identifies a Population With Greater Risk of Disease Recurrence When Treated With Endocrine Therapy Alone.

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 of >90% 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.

Proteomic characterisations of ulcerative colitis endoscopic biopsies associate with clinically relevant histological measurements of disease severity.

AIMS AND METHODS: Accurate 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. RESULTS: Targeted 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 ß-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. CONCLUSIONS: Pathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

Novel ophthalmic pathology in an autopsy case of autosomal dominant retinal vasculopathy with cerebral leukodystrophy.

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.

Multiplex Quantitative Analysis of Tumor-Infiltrating Lymphocytes, Cancer-Associated Fibroblasts, and CD200 in Pancreatic Cancer.

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.

Quantitative Assessment of CD200 and CD200R Expression in Lung Cancer.

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 < 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.

Analysis of Immune Checkpoint Drug Targets and Tumor Proteotypes in Non-Small Cell Lung Cancer.

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.

A novel immunohistochemical scoring system reveals associations of C-terminal MET, ectodomain shedding, and loss of E-cadherin with poor prognosis in oral squamous cell carcinoma.

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.

A Randomized-Controlled Phase 2 Study of the MET Antibody Emibetuzumab in Combination with Erlotinib as First-Line Treatment for EGFR Mutation-Positive NSCLC Patients.

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.

The interaction profile of homologous recombination repair proteins RAD51C, RAD51D and XRCC2 as determined by proteomic analysis.

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.

Functional characterization and identification of mouse Rad51d splice variants.

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 RAD51DDelta7b (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 Rad51dDelta3 (deleted for exon 3) and Rad51dDelta5 (deleted for exon 5)transcripts display tissue specific expression patterns with Rad51dDelta3 being detected in each tissue except ovary and Rad51dDelta5 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.

Extensive chromosomal instability in Rad51d-deficient mouse cells.

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.

Analysis of Peripheral T-cell Lymphoma Diagnostic Workup in the United States.

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.