Paraffin-embedded Biopsy Specimens Research Articles

Matrix Metalloproteinase-9 Expression and Status of Cervical Lymph Nodes in Patients with Nasopharyngeal Carcinoma.

Tumor growth and metastasis in nasopharyngeal carcinoma (NPC) patients was suspected as a role of several molecular biomarkers that have been identified in tumor specimens of patients with NPC. Invasion and metastasis process was a complex mechanism which involved some proteolytic enzymes, such as matrix metalloproteinase-9 (MMP-9). To analyze the association of MMP-9 expression of NPC patients with cervical lymph node metastasis. The study was conducted in oncology unit of ORL-HNS at Dr. Soetomo General Hospital Surabaya from May to July 2015. Formalin-fixed paraffin-embedded biopsy specimens from NPC patients with WHO type II and III of histopathology and clinically were divided into four state of cervical enlargement (N0. N1. N2 and N3). The expression of MMP-9 was obtained with immunohistochemistry using rabbit polyclonal antibody Anti-MMP9 ab7299 from abcam®. Cambridge-UK. Thirty-two NPC patients were enrolled in this study. The study found a negative expression of MMP-9 in 3.12% of samples. Spearman rho test result was p = 0.001 with correlation coefficient of 0.754. Spearman test resulted p value of 0.001 with a correlation coefficient of 0.754. Correlation of matrix metalloproteinase-9 expression with cervical lymph node metastasis (N0, N1, N2, and N3) in patients with NPC showed a significant result (p < 0.05). There was a strong positive correlation between MMP-9 expressions with cervical lymph node status in NPC patients.

Corynebacterium kroppenstedtii in granulomatous mastitis: Analysis of formalin-fixed, paraffin-embedded biopsy specimens by immunostaining using low-specificity bacterial antisera and real-time polymerase chain reaction.

Granulomatous mastitis (GM) is a rare inflammatory disease of the post-lactation breast, clinically mimicking breast cancer. GM is microscopically characterized by formation of epithelioid granulomas and abscess (suppurative granulomas) with lipid droplet-centered inflammation. Corynebacterium kroppenstedtii (Ck) is known as a causative bacterium of GM, and identification of Ck infection within the lesion should thus be essential for confirming the diagnosis. In the present study, we analyzed formalin-fixed, paraffin-embedded (FFPE) biopsy specimens of a total of 18 GM lesions with immunostaining and real-time PCR for Ck genome. Widely cross-reactive rabbit antisera against Bacillus Calmette-Guerin (BCG), Bacillus cereus, Treponema pallidum and Escherichia coli were chosen. With real-time PCR, Ck genome was demonstrated in 7 of 18 GM lesions. Immunohistochemically, the low-specificity antisera reacted with the cytoplasm of phagocytes and/or granuloma-engulfed lipid droplets in 12 of 18 GM lesions. Antigenic positivity was observed in the following order: BCG > B. cereus > T. pallidum > E. coli. Real-time PCR using DNA extracted from FFPE sections was useful but not consistent for identifying the Ck genome in GM, while immunostaining using cross-reactive antisera against four kinds of bacteria was not Ck-specific but was applicable to visualizing bacterial infection within the GM lesions.

The Amsterdam ReBus progressor cohort: identification of 165 Barrett's surveillance patients who progressed to early neoplasia and 723 nonprogressor patients.

Patient selection is suboptimal in most studies focused on identifying biological markers for neoplastic progression in Barrett's esophagus (BE). This study aims to describe a stringently selected community-based case-control cohort of non-dysplastic BE (NDBE) patients who progressed to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC) and BE patients who never progressed to be used for future biomarker studies. We identified all patients referred for endoscopic work-up of BE neoplasia at three tertiary referral centers for treatment of BE neoplasia between 2000 and 2013. We performed a detailed registration of any endoscopic surveillance history before neoplastic progression. Controls were selected from a retrospective BE surveillance registration in 10 community hospitals. A total of 887 patients were referred for endoscopic work-up of BE neoplasia. Based on predefined selection criteria, we identified 165 progressor patients (82% men; mean age 55 years ± 10.4) with a baseline endoscopy demonstrating NDBE > 2 years before neoplastic progression. Using the same predefined selection criteria, 723 nonprogressor patients (67% men; mean age 57 years ± 11.3) with >2 years of endoscopic surveillance were identified. Median length of the BE segment was 5 cm (IQR 4-7) in progressors and 4 cm (IQR 2-6) in controls. Median duration of surveillance was 89 months (IQR 54-139) in progressors and 76 months (IQR 47-116) in nonprogressors. Paraffin embedded biopsies are available for biomarker research in all patients. Ethical approval was obtained and material transfer agreements were signed with all 58 contributing pathology labs. This is the largest community-based case-control cohort of BE patients with and without progression to early neoplasia. The stringent selection criteria and the availability of paraffin embedded biopsy specimens make this a unique cohort for biomarker studies.

Integrated DNA/RNA targeted genomic profiling of diffuse large B-cell lymphoma using a clinical assay

We sought to define the genomic landscape of diffuse large B-cell lymphoma (DLBCL) by using formalin-fixed paraffin-embedded (FFPE) biopsy specimens. We used targeted sequencing of genes altered in hematologic malignancies, including DNA coding sequence for 405 genes, noncoding sequence for 31 genes, and RNA coding sequence for 265 genes (FoundationOne-Heme). Short variants, rearrangements, and copy number alterations were determined. We studied 198 samples (114 de novo, 58 previously treated, and 26 large-cell transformation from follicular lymphoma). Median number of GAs per case was 6, with 97% of patients harboring at least one alteration. Recurrent GAs were detected in genes with established roles in DLBCL pathogenesis (e.g. MYD88, CREBBP, CD79B, EZH2), as well as notable differences compared to prior studies such as inactivating mutations in TET2 (5%). Less common GAs identified potential targets for approved or investigational therapies, including BRAF, CD274 (PD-L1), IDH2, and JAK1/2. TP53 mutations were more frequently observed in relapsed/refractory DLBCL, and predicted for lack of response to first-line chemotherapy, identifying a subset of patients that could be prioritized for novel therapies. Overall, 90% (n = 169) of the patients harbored a GA which could be explored for therapeutic intervention, with 54% (n = 107) harboring more than one putative target.

Intracell Heat Shock Protein 70 Expression and Nasopharyngeal Carcinoma Stage.

Tumor growth and metastasis in nasopharyngeal carcinoma (NPC) patients recent research stated that intracellular Hsp70 is a stress protein as the main factor for the growth, invasion and metastasis of NPC. To analyze intracellular Hsp70 protein expression of NPC patients which correlate with staging of NPC as clinical manifestation. Formalin-fixed paraffin-embedded biopsy specimens from 20 NPCs histopathology and clinically. The expression of intracellular Hsp70 was obtained using monoclonal antibody of Anti Human Hsp70 from Santa Cruz Biotechnology, California, USA. The assessment of the staining was performed with Remmele methode by Histopathologies doctor Consultant. 20 NPC patients met the inclusion and exclusion criteria. The data showed a negative intracellular of Hsp70 expression by 15% for all sample that consist of 1 patient of stage I, II, III, and none for stage IV. Then, followed by 25% of mild positive intracellular Hsp70 expression which consisted of 0 patient of stage I and III, 2 patients of stage II, and 3 patients of stage IV. Followed by 50% of moderate positive intracellular Hsp70 expression which consisted of 0 patient of stage I and II, 1 patient of stage III, and 9 patients of stage IV. Spearman test results test scored p = 0.001 with a correlation coefficient of 0.671. The correlation of intracellular Hsp70 protein expression with stage (I, II, III, and IV) in NPC patients was significant (p < 0.05). There was a correlation between increased intracellular Hsp70 expression and the stage of patients with nasopharyngeal carcinoma.

Abstract P2-03-02: Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer

Abstract Background: An on-demand, closed system RT-qPCR (the GeneXpert system, Cepheid, Sunnyvale, CA) has the potential to provide biomarker information in low resourced settings. The system consists of an inexpensive, single-use, disposable, macrofluidic cartridge and an instrument that automates RT-qPCR. Here we use it with a research use only cartridge (STRAT4) that measures the mRNA expression levels of ESR1, PGR, ERBB2, and MKi67 using a single 5uM thick FFPE tissue section from an excisional or core biopsy specimen containing invasive carcinoma of the breast. The assay, results are expressed as a delta cycle threshold (dCt) value, defined as the Ct of a control gene (CYFIP1) minus the Ct of the target gene (ESR1, PGR, ERBB2, or MKi67). We determine whether the dCt result for each marker is equivalent using the entire non-macrodissected section (non m-d) to the dCt results obtained following tumor macro-dissection (m-d) to eliminate non-tumor elements from the assay. Methods: We evaluated the impact of m-d versus non m-d using STRAT4 on a cohort of 62 formalin-fixed paraffin-embedded (FFPE) tumor core needle biopsy specimens with a range of HER2 expression determined by clinical immunohistochemistry and fluorescence in situ hybridization (IHC/FISH). Concordance (sensitivity and specificity) of the STRAT4 ESR1 and HER2 mRNA versus ER and HER2 IHC/FISH measurements were also assessed. Results: We observed excellent agreement of the resulting dCt between the paired samples, m-d versus non m-d, for ESR1 (R2=0.92), PGR (R2=0.90), ERBB2 (R2=0.94) and MKi67 (R2=0.90). No significant difference (P value &amp;gt; 0.99) was observed when we compared the dCt between the paired samples m-d versus non m-d. In addition, using the predefined STRAT4 dCt cutoff for ESR and ERBB2 positivity, we found a significant concordance between RT-qPCR and IHC/FISH for ESR-positivity for the paired samples, m-d (P value &amp;lt; 0.001; sensitivity = 0.98; specificity = 1; PPV = 1; NPV = 0.95) versus non m-d (P value &amp;lt; 0.001; sensitivity = 0.98; specificity = 1; PPV = 1; NPV = 0.95) and HER2-positivity for the paired samples, m-d (P value &amp;lt; 0.001; sensitivity = 0.85; specificity = 0.98; PPV = 0.92; NPV = 0.96) versus non m-d (P value &amp;lt; 0.001; sensitivity = 0.71; specificity = 0.98; PPV = 0.90; NPV = 0.92), respectively. Conclusion: These data suggest that mRNA for ESR and ERBB2 is sufficiently low in surrounding tissues that m-d of whole sections is not required for accurate assessment of key breast cancer mRNA markers in a closed system RT-qPCR assay. The simplicity of the assay workflow may be particularly valuable in low resourced settings where routine access to pathology expertise and to high quality IHC/FISH is challenging. Citation Format: Gupta S, Carvajal-Hausdorf DE, Wasserman BE, Ho K, Weidler J, Wong W, Rhees B, Bates M, Rimm DL. Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-02.

Expression of Nedd9 in Transbronchial Biopsies of Lung Adenocarcinoma.

SUMMARY – Lung cancer as the major cause of cancer mortality worldwide includes several histologic subtypes evolving from numerous genetic and epigenetic changes emerging in alveolar, bronchiolar and bronchial epithelium. The majority of cases are diagnosed in advanced stage (i.e. stages IIIB and IV), often with scanty amount of tissue in transbronchial biopsies or cytology specimens. The aim of the present study was to investigate the expression of the scaffolding protein neural precursor cell expressed, developmentally downregulated 9 (NEDD9) in small biopsies of lung adenocarcinoma. The expression of NEDD9 was analyzed immunohistochemically in 71 formalin-fixed and paraffin-embedded transbronchial biopsy specimens of primary lung adenocarcinoma. Nuclear and cytoplasmic NEDD9 expression was detected indicating activation of the epithelial-mesenchymal transition process. Direct relationship between the expression of NEDD9 and survival was not demonstrated.

DnaJ Homolog Subfamily B Member 9 Is a Putative Autoantigen in Fibrillary GN.

Fibrillary GN is a rare form of GN of uncertain pathogenesis that is characterized by the glomerular accumulation of randomly arranged, nonbranching fibrils (12-24 nm) composed of Ig and complement proteins. In this study, we used mass spectrometry to comprehensively define the glomerular proteome in fibrillary GN compared with that in controls and nonfibrillary GN renal diseases. We isolated glomeruli from formalin-fixed and paraffin-embedded biopsy specimens using laser capture microdissection and analyzed them with liquid chromatography and data-dependent tandem mass spectrometry. These studies identified DnaJ homolog subfamily B member 9 (DNAJB9) as a highly sampled protein detected only in fibrillary GN cases. The glomerular proteome of fibrillary GN cases also contained IgG1 as the dominant Ig and proteins of the classic complement pathway. In fibrillary GN specimens only, immunofluorescence and immunohistochemistry with an anti-DNAJB9 antibody showed strong and specific staining of the glomerular tufts in a distribution that mimicked that of the immune deposits. Our results identify DNAJB9 as a putative autoantigen in fibrillary GN and suggest IgG1 and classic complement effector pathways as likely mediators of the destructive glomerular injury in this disease.

CORRELATION BETWEEN INTRACELLULAR HEAT SHOCK PROTEIN 70 EXPRESSION AND CERVICAL LYMPH NODES ENLARGEMENT IN NASOPHARYNGEAL CARCINOMA

Tumor growth and metastasis in NPC patients suspected role of several molecular biomarkers that have been identified in tumor specimens of patients with NPC. Recent research states intracellular Hsp70 is a stress protein that is a main factor for the growth, invasion and metastasis of NPC. The purpose of this study is to analyze intracellular Hsp70 protein expression of NPC patients which correlate with staging of NPC as clinical manifestation. This study used cross sectional design. In this study, formalin-fixed paraffin-embedded biopsy specimens were obtained from 18 NPCs with all of histopathology and clinically divided into N0, N1, N2, N3. The expression of intracellular Hsp70 was obtained with immunohistochemistry using monoclonal antibody Anti Human Hsp70 antibodi from Santa Cruz Biotechnology, California, USA. Assessment of the staining was performed with Remmele methode by Histopathologies doctor Consultant. The Spearman’s test was used to determine the relationship between expression of intracellular Hsp70 protein and enlargement cervical nodul of NPC. Statistical significance was defined as p &lt;0,05. There were 18 patients NPC patients that met the inclusion and exclusion criteria. Showed negative intracellular Hsp70 expression by 16,67% for all sample in which consist 1 patient of N0, 1 patient of N1, 1 patient of N2, 0 patient of N3. Followed by 27,78% mild positive intracellular Hsp70 expression which consist 0 patient of N0, 2 patients of N1, 0 patient of N2, 3 patients of N3. Followed by 44,44% moderate positive intracellular Hsp70 expression which consist 0 patient of N0, 0 patient of N1, 1 patient of N2, 9 patients of N3. Followed by 11,11% strongly positive intracellular Hsp70 expression which consist 0 patient of N0, 0 patient of N1, 0 patient of N2, 2 patients of N3. Spearman test results test scores p = 0.01 with a correlation coefficient 0.606. Correlation of intracellular Hsp70 protein expression with enlargement cervical nodul of lymphe (N0, N1, N2, N3) in patients with NPC showed significant (p&lt;0.05). In conclusion, there was correlation between increase of the intacellular Hsp70 expression and enlargement cervical nodul of lymphe in nasopharyngeal carcinoma.

Fluorescence In Situ Hybridization for Diagnosis of Whipple's Disease in Formalin-Fixed Paraffin-Embedded Tissue.

Whipple’s disease (WD) is a rare chronic systemic infection with a wide range of clinical symptoms, routinely diagnosed in biopsies from the small intestine and other tissues by periodic acid–Schiff (PAS) diastase staining and immunohistological analysis with specific antibodies. The aim of our study was to improve the pathological diagnosis of WD. Therefore, we analyzed the potential of fluorescence in situ hybridization (FISH) for diagnosing WD, using a Tropheryma (T.) whipplei-specific probe. 19 formalin-fixed paraffin-embedded (FFPE) duodenal biopsy specimens of 12 patients with treated (6/12) and untreated (6/12) WD were retrospectively examined using PAS diastase staining, immunohistochemistry, and FISH. 20 biopsy specimens with normal intestinal mucosa, Helicobacter pylori, or mycobacterial infection, respectively, served as controls. We successfully detected T. whipplei in tissue biopsies with a sensitivity of 83% in untreated (5/6) and 40% in treated (4/10) cases of WD. In our study, we show that FISH-based diagnosis of individual vital T. whipplei in FFPE specimens is feasible and can be considered as ancillary diagnostic tool for the diagnosis of WD in FFPE material. We show that FISH not only detect active WD but also be helpful as an indicator for the efficiency of antibiotic treatment and for detection of recurrence of disease when the signal of PAS diastase and immunohistochemistry lags behind the recurrence of disease, especially if the clinical course of the patient and antimicrobial treatment is considered.

Multiplex Detection of Pediatric Low-Grade Glioma Signature Fusion Transcripts and Duplications Using the NanoString nCounter System.

Previous studies identified recurrent fusion and duplication events in pediatric low-grade glioma (pLGG). In addition to their role in diagnosis, the presence of these events aid in dictating therapy and predicting patient survival. Clinically, BRAF alterations are most commonly identified using fluorescent in situ hybridization (FISH). However, this method is costly, labor-intensive and does not identify nonBRAF events. Here, we evaluated the NanoString nCounter gene expression system for detecting 32 of the most commonly reported fusion/duplication events in pLGG. The assay was validated on 90 pLGG samples using FISH as the gold standard and showed sensitivity and specificity of 97% and 98%, respectively. We next profiled formalin-fixed paraffin-embedded preserved biopsy specimens from 429 pLGG cases. 171 (40%) of the cases within our cohort tested positive for a fusion or duplication event contained within our panel. These events, in order of prevalence, were KIAA1549-BRAF 16;9 (89/171, 52.0%), KIAA1549-BRAF 15;9 (42/171, 24.6%), KIAA1549-BRAF 16;11 (14/171, 8.2%), FGFR1-TACC1 17;7 (13/171, 7.6%), MYBL1 duplication (5/171, 2.9%), KIAA1549-BRAF 18;10 (4/171, 2.3%), KIAA1549-BRAF 15;11 (2/171, 1.2%), FAM131B-BRAF 2;9 (1/171, 0.6%), and RNF130-BRAF 3;9 (1/171, 0.6%). This work introduces NanoString as a viable clinical replacement for the detection of fusion and duplication events in pLGG.

Low Expression of Estrogen Receptor-α and Progesterone Receptor in Human Breast Cancer Tissues Is Associated With High-Grade Human Cytomegalovirus Protein Expression

BackgroundThe underlying mechanisms for breast cancer (BC) are largely unknown. We investigated possible correlations between the expression levels of human cytomegalovirus (HCMV) proteins and established histopathological markers of BC, including expression of estrogen receptor (ER)-α, the progesterone receptor (PgR), and HER2. Materials and MethodsWe retrospectively examined paraffin-embedded biopsy specimens of BC (n = 62), ductal carcinoma in situ (n = 19), and adjacent normal breast tissue (n = 42) for HCMV immediate-early protein (IE), HCMV late antigen, HCMV DNA and RNA, and investigated possible correlations between them and expression of ER-α, PgR, and HER2. ResultsHCMV DNA and RNA were detected in all examined infiltrating BCs. High-grade positivity for HCMV-IE was detected in 77% of infiltrating BCs, 39% of ductal carcinomas in situ, and 7% of tumor-free breast tissue samples. HCMV expression correlated inversely with ER-α (P = .02) and PgR (P = .003) expression. HER2 expression was also reduced in HCMV-positive samples without reaching a level of statistical significance (P = .09). ConclusionThe negative correlation between high-grade expression HCMV-IE and hormone receptor expression suggests a role for HCMV in hormone receptor-negative BC tumors, possibly by forcing BC cells into a more aggressive phenotype.

Patient selection using novel RNA-based approaches: Early experience from a phase I study with the pan-FGFR inhibitor BAY 1163877 in patients with urothelial bladder cancer.

386 Background: FGFRs play a role in a variety of malignancies, including urothelial bladder cancer (UBC). Whereas genetic alterations of FGFRs in UBC are known, non-genetic and epigenetic activation of FGFR gene expression have also been described. BAY 1163877 is an oral inhibitor of FGFRs 1-3 with antitumor activity in FGFR mRNA overexpressing xenograft models not limited to DNA alterations. We report the results from a phase I expansion cohort in UBC patients pre-screened for treatment with BAY 1163877 based on FGFR1-3 mRNA overexpression and/or activating mutations in the FGFR3 gene. Methods: FGFR mRNA levels were assessed in paraffin-embedded archival or fresh biopsy specimens by RNAscope and NanoString technology and activating mutations by RT-PCR-based mutation assay. Tumor response to treatment was assessed by RECIST, v1.1. Adverse events were assessed using CTCAE v4.03 criteria. Results: A total of 80 patients from multiple indications were enrolled in the phase I study; 800 mg BID was established as the recommended phase 2 dose. Sixty seven UBC patients were screened, with 31 tumor biopsies (46%) determined to overexpress FGFR mRNA: 2 patients each with FGFR1 and FGFR2, and 27 patients with FGFR3. Of the 27 patients with FGFR3 mRNA overexpression, 5 had in parallel an activating mutation in FGFR3 gene. Eight UBC patients met eligibility criteria and entered treatment; all had elevated FGFR3 mRNA expression and 4 had alterations in FGFR3 DNA (3 mutations and 1 translocation). Seven patients (87.5%) had tumor shrinkage in target lesions as best response, with 3/8 partial responses (PR; 37.5%). One patient with a PR presented with elevated FGFR3 mRNA without evidence of aberrant DNA. BAY 1163877 was generally well tolerated and AEs manageable with dose modification. The most common AEs in the overall population were increased phosphorus (66% all grade, 1 grade-3 event) and diarrhea (33% all grade, 2 grade-3 events). Conclusions: Selection of patients for treatment with BAY 1163877 based on FGFR mRNA expression levels in archival tissue was feasible. BAY 1163877 had a favorable safety profile and promising anti-tumor activity in UBC patients. Clinical trial information: NCT01976741.

Molecular Profiling to Determine Clonality of Serial Magnetic Resonance Imaging/Ultrasound Fusion Biopsies from Men on Active Surveillance for Low-Risk Prostate Cancer

Purpose: To determine whether MRI/ultrasound (MRI/US) fusion biopsy facilitates longitudinal resampling of the same clonal focus of prostate cancer and to determine whether high-grade cancers can evolve from low-grade clones.Experimental Design: All men on active surveillance who underwent tracking MRI/US fusion biopsy of Gleason 6 prostate cancer, on at least two distinct occasions, between 2012 and 2014 were enrolled. MRI/US fusion was used to track and resample specific cancer foci. IHC for ERG and targeted RNA/DNA next-generation sequencing (NGS) were performed on formalin-fixed paraffin-embedded prostate biopsy specimens to assess clonality.Results: Thirty-one men with median age and PSA of 65 years and 4.6 ng/mL, respectively, were analyzed. The median sampling interval was 12 months (range, 5-35). Of the 26 evaluable men, ERG IHC concordance was found between initial and repeat biopsies in 25 (96%), indicating resampling of the same clonal focus over time. Targeted NGS supported ERG IHC results and identified unique and shared driving mutations, such as IDH1 and SPOP, in paired specimens. Of the nine men (34.6%) who were found to have Gleason ≥7 on repeat biopsy, all displayed temporal ERG concordance. Prioritized genetic alterations were detected in 50% (13/26) of paired samples. Oncogenic mutations were detected in 22% (2/9) of Gleason 6 cancers prior to progression and 44% (4/9) of Gleason ≥7 cancers when progression occurred.Conclusions: Precise tracking of prostate cancer foci via MRI/US fusion biopsy allowed subsequent resampling of the same clonal focus of cancer over time. Further research is needed to clarify the grade progression potential of Gleason 6 prostate cancer. Clin Cancer Res; 23(4); 985-91. ©2016 AACR.

Immunohistochemical expression of Survivin in oral leukoplakia and oral squamous cell carcinoma

Background and Objectives: Survivin is an inhibitor of apoptosis protein. It is involved in the modulation of the cell death and cell division process. Alterations in the expression of Survivin have been reported in several inflammatory, premalignant, and malignant lesions. Hence, the objectives of the present study were to compare the expression of Survivin with respect to the degree of dysplasia in oral leukoplakia (OL) and to evaluate the expression of Survivin with respect to different histopathological grades of oral squamous cell carcinoma (OSCC). Materials and Methods: Neutral-buffered, formalin-fixed and paraffin-embedded biopsy specimens from 30 patients, each with OL and OSCC, were included in this study. The expression of Survivin was detected by immunohistochemistry. The scores obtained were subjected to Chi-square test. Results: Survivin expression was detected in all grades of dysplasia and OSCC. Expression of Survivin was significant when compared with different degrees of dysplasia and different histopathological grades of OSCC (P Conclusion: Higher immunohistochemical scores were obtained with increased histopathological grades of OL and OSCC. High expression of Survivin may be related to malignant transformation in OL and poor prognosis in OSCC.

Multiplex Polymerase Chain Reaction for the Detection of High-risk-Human Papillomavirus Types in Formalin-fixed Paraffin-embedded Cervical Tissues

Detecting high-risk-human papillomavirus (HPV) types has become an integral part of the cervical cancer screening programmes. This study aimed to develop a multiplex polymerase chain reaction (PCR) for identification of HPV types 16 and 18 along with the beta globin gene in formalin-fixed and paraffin-embedded cervical biopsy specimens. A total of 59 samples from patients with cervical abnormalities were tested. HPV 16 positivity was 50% in cervical cancers and 52.9% in cervical intraepithelial neoplasia. Our multiplex PCR protocol can be used as a simple and cost-effective tool for high-risk-HPV detection in cervical cancer screening programmes.

Multiplex Quantitative Imaging Analysis of Bone-Remodeling Cells in Patients with Myelofibrosis

Background: Bone marrow (BM) fibrosis is a major cause of morbidity and mortality in patients with myeloproliferative neoplasms. Its progression correlates with thickening and distortion of the bone trabeculae, a phenomenon commonly referred to as osteosclerosis. The physiological process of bone remodeling depends on a delicate interplay between monocyte-derived bone-resorbing osteoclasts and mesenchymal cell-derived bone-forming osteoblasts. Whereas impairment in bone resorption is believed to be the underlying cause of inherited sclerosing bone dysplasias, little is known about the pathogenesis of osteosclerosis associated with myelofibrosis (MF). Phenotypically complex bone-remodeling cells are attached to the bone surface and as such are difficult to study by conventional techniques. Here, we present a novel method for visualization and quantification of these cells in situ and assess their role in inducing osteosclerotic changes in the BM of patients with MF.Methods: Formalin-fixed paraffin-embedded BM biopsy specimens were obtained from the iliac crest of patients with advanced stages of MF (MF-2 and MF-3) and healthy controls. Staining of each sample was performed using a series of previously validated monoclonal antibodies directed against osteoclast- (TRAP and cathepsin K) and osteoblast-specific antigens (CD56 and RUNX2). To achieve non-interfering identification, we employed a tyramide-based fluorescent immunohistochemistry approach (PerkinElmer Opal). Specimens were imaged in toto using an automated multispectral system at 0.5 µm resolution and segmented to individual cells via a pattern recognition algorithm. Cells were assigned to one of the two phenotypes based on specific signal intensities and size. Finally, cells lying within 20 µm of the bone perimeter were quantified utilizing methods of spatial statistics.Results: Samples from 10 MF patients (age 63 ± 8 years, median ± MAD) were compared to 3 healthy controls (age 48 ± 7 years). Area of examined tissue ranged from 8.2 to 38.4 mm2 with the median BM to bone ratio of 1.6:1 and 3.5:1, in MF and controls respectively. Discrimination of bone-remodeling subsets by the assay demonstrated high sensitivity and specificity, estimated both visually (Figure 1) and quantitatively. In MF patients we detected 7.1 ± 3.1 osteoclasts per 100 mm of bone perimeter (mean ± SEM) versus 2.4 ± 1.9 per 100 mm in healthy controls (p < 0.0001). Frequency of observable osteoblasts per bone perimeter also showed a significant increase in MF compared to healthy controls (p < 0.0001). Quantification using area of the bone as denominator was in concordance with the densities found per bone perimeter and similar statistical significance was observed.Conclusions: Our analytical method offers a feasible approach for in situ investigation of complex cellular subsets in the BM. This analysis demonstrates that both osteoclasts and osteoblasts are pathologically affected in advanced stages of MF. Expansion of osteoblastic population supports the involvement of cells of mesenchymal lineage in the progression of osteosclerosis. By contrast, an overwhelmingly increased density of osteoclasts suggests a functional loss of these cells of monocytic origin. This is particularly intriguing in view of the emerging role neoplastic monocyte-derived fibrocytes play in the pathogenesis of MF. Further studies exploiting the interaction between these cells will likely shed light on the pathogenesis of osteosclerosis in MF. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical and Prognostic Significance of Epstein-Barr Virus in Diffuse Large B-Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the largest subtype of lymphoma, but consists of heterogeneous groups with different prognosis. Many studies conducted to determine prognosis factors of DLBCL. Recently, several studies have been reported that the elderly Epstein-Barr virus (EBV)-positive DLBCL patients showed worse prognosis than the elderly EBV-negative DLBCL. (Oyama T et al. Clin Cancer Res 2007;13:5024-5032). On the other hand, other studies reported that EBV-positivity is not a prognosis factor in young-adult DLBCL patients (Hong JY et al. Annals of Oncology 2015;26:548-555, Nicolae A et al. BLOOD 2015;126:863-872). In the present study, retrospective analyses were carried out for patients with DLBCL diagnosed between 1990 and 2007 to analyze the clinical and prognostic significance of EBV.Patients: Those who met the following criteria were included in the study: (i) pathology confirmed de novo DLBCL, according to the WHO classification; (ii) adequate amount and quality of paraffin-embedded biopsy specimens or unstained slides for EBV-encoded RNA in situ hybridization.Statistical methods: Intergroup comparisons were carried out with Fisher's exact test for categorical variables and the Mann-Whitney test for age. For survival analysis, we carried out Kaplan-Meier and multivariate proportional hazard (Cox) analyses. Statistical analyses were carried out using the software Stata SE version 14.0.Results: A total of 456 patients were included. The median follow up duration of survivor was 100 months (range, 1.9-363). There were 236 male (51.75%) and 220 female patients (48.25%) with a median age of 67 (range, 22-94) years. 246 patients (54.0%) were in advanced (III/IV) stage, 134 (29.4%) presented with poor Eastern Cooperative Oncology Group Performance Status (ECOG-PS), 275 (60.3%) with elevated lactate dehydrogenase (LDH) level, 263 (57.7%) with extranodal involvement of 1 or more sites, and 64 (14.0%) with bulky mass. As a consequence, 211 patients (46.3%) were in high/high-intermediate International Prognostic Index (IPI) categories. 5 years overall survival (OS) was 66.2%, in total. EBV was detected in samples from 47 patients (10.3 %). The positivity was 10.6% (15 / 142) in patients of 60 years or younger, and 10.2 % (32 / 314) in those older than 60 years (p = 0.51). No differences in back ground date were found between EBV - positive and negative DLBCLs regarding age (median 66.5 vs. 64.8, P = 0.17), male sex (88.1% vs. 11.9%, P = 0.16), advanced stage (88.2% vs. 11.8%, P = 0.17), poor ECOG-PS (90.3% vs. 9.7%, P = 0.47), elevated LDH (89.5% vs. 10.6%, P = 0.48), extranodal sites of one or more (90.5% vs. 9.5%, P = 0.31), bulky mass (92.2% vs. 7.8%, P = 0.33), and higher IPI categories (88.6% vs. 11.4%, P = 0.29).The prognosis was also not different between EBV-positive and negative DLBCLs. The 5 years OS was 42.6% for EBV-positive DLBCLs, and 56.4% for EBV-negative DLBCLs (P = 0.11). The same results were found for the younger (53.3% vs. 66.9%, P = 0.30) and the elderly patients (42.9% vs. 51.8%, P = 0.18).Conclusion: In this practice based, uncontrolled study, EBV-positively wad not different in younger and elderly DLBCLs. Although EBV-positive DLBCL showed worse trend of prognosis, the difference was not statistically significant. The significance of EBV in DLBCL should be reconsidered in unbiased, large-scale patient date. [Display omitted] [Display omitted] [Display omitted] DisclosuresSuzuki:Chugai: Honoraria; Bristol-Myers Squibb: Honoraria; Kyowa Hakko kirin: Honoraria. Suzumiya:Chugai: Research Funding, Speakers Bureau; Astellas: Research Funding; Toyama Chemical: Research Funding; Eizai: Research Funding; Takeda: Speakers Bureau; Kyowa Hakko Kirin: Research Funding.

Abstract 4027: Recurrent gains of CD274 (PD-L1) and PDCD1LG2 (PD-L2) provide a genetic basis for PD-1 ligand expression in a subset of solid tumors

Abstract Background: A subset of malignant tumors are immunogenic but avoid immune surveillance through ectopic expression of the programmed cell death 1 ligands, PD-L1 and PD-L2, that bind PD-1 on effector T-cells to inhibit T-cell activation, a phenotype that is reversible with PD-1 blockade. Co-amplification and co-gain of CD274 (PD-L1) and PDCD1LG2 (PD-L2) on chromosome 9p24.1 provide a genetic basis for PD-1 ligand expression by Reed-Sternberg cells in classical Hodgkin lymphoma, a tumor highly sensitive to PD-1 blockade. We sought to determine whether copy number alterations of CD274 and PDCD1LG2 are a mechanism of PD-1 ligand expression in other tumor types. Methods: We surveyed data derived from a clinically-deployed next generation sequencing assay (OncoPanel) to identify tumors with evidence of selective copy gain of genes located at 9p24.1. Formalin-fixed paraffin-embedded biopsy specimens from available cases were reviewed for tumor content, immunostained for PD-L1, and analyzed with custom probes targeting the CD274 and PDCD1LG2 loci and a commercial probe targeting the centromere of chromosome 9 by flourescence in situ hybridization (FISH). Results: Review of NGS data revealed 171 of 7,070 clinical samples (2.4%) with evidence for selective copy gain of CD274 and/or PDCD1LG2. Tissue was available for analysis in 73 cases, of which 55 were successfully analyzed. Co-amplification and co-gain of CD274 and PDCD1LG2 were observed for 20 (36%) and 18 (33%) of cases, respectively. Polysomy 9 and disomy 9 were observed among 9 (16%) and 2 (4%) cases, respectively. Genetic abnormalities associated with chromosome 9 loss were observed among the remaining cases 6 (11%). PD-L1 IHC revealed positive staining of tumor cells in 36 (65%) cases. Among cases with co-amplification/gain of CD274 and PDCD1LG2, a subset (Co-Amp: 36%; Co-Gain: 28%) exhibited moderate to high PD-L1 expression in significant percentages of tumor cells (Tumor H-score &amp;gt; 50). Tumor types with high PD-L1 expression in the context of co-amplification/gain included carcinomas of the head and neck, bladder, and cervix. Conclusions: Review of over 7000 tumors analyzed by a clinically-deployed NGS platform identifies CD274 and PDCD1LG2 copy gain in a low percentage of a variety of solid tumor types, a subset of which has a corresponding increase of PD-L1 expression. These data suggest that copy gain of CD274 and PDCD1LG2 contributes to PD-1 ligand expression in a subset of solid tumors beyond classical Hodgkin lymphoma. Citation Format: Evisa Gjini, Christine Pak, Alyssa Kelly, Yuling Shi, Neal Lindeman, Frank Kuo, Gordon Freeman, Azra Ligon, Stephen Hodi, Margaret Shipp, Scott Rodig. Recurrent gains of CD274 (PD-L1) and PDCD1LG2 (PD-L2) provide a genetic basis for PD-1 ligand expression in a subset of solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4027.

Molecular and metabolic differences of treatment responders versus nonresponders in a phase 0 clinical trial of metformin in endometrial cancer

Objectives: In a phase 0 clinical trial of metformin in endometrial cancer (EC) patients, we previously reported that metformin decreased proliferation in the tumors of the majority of patients enrolled. We aimed to determine molecular and metabolic differences between responders and nonresponders to metformin treatment. Methods: Obese women with endometrioid EC were enrolled. Patients took metformin (850 mg QD) for 1 to 4 weeks before surgical staging. Tissue microarrays (triplicate cores) were constructed from formalin-fixed, paraffin-embedded endometrial biopsy and hysterectomy specimens before and after metformin treatment. Expression of the metformin transporters (OCT1, OCT3, PMAT, MATE1, MATE2), phosphorylated (p)-insulinlike growth factor 1 receptor (IGF1R), p-insulin receptor substrate-1 (IRS1), LKB1, and PTEN were evaluated using immunohistochemistry. Global, untargeted metabolomics was performed on serum samples of EC patients before metformin treatment, using combined GC-MS and LC-MS/MS techniques. Results: Of 20 patients enrolled, 13 (65%) responded to metformin as defined by a significant decrease in Ki-67 staining. In responders, metformin decreased the expression of OCT1 (46%, P = .003), OCT3 (40%, P = .036), PMAT (66%, P = .0019), MATE2 (26%, P = .0017), p-IGF1R (24%, P = .035), LKB1 (36%, P = 0.0015), and PTEN (80%, P = .033) but was not associated with changes in MATE1 or p-IRS1. In nonresponders, metformin treatment was not associated with any changes in these molecular targets. Pretreatment MATE2 expression approached significance in predicting response to metformin (P = .0625). Metabolomic profiling revealed that responders had higher premetformin treatment levels of amino acids, dipeptides, glycolytic intermediates, arachidonic acid, monohydroxy fatty acids and lysolipids compared with nonresponders (P < .05). Conclusions: Treatment response to metformin was associated with decreased expression of OCT1, OCT3, PMAT, and MATE2 transporters as well as metabolically relevant proteins associated with obesity and EC. Baseline metabolic differences were found between responders and nonresponders to metformin that may serve as biomarkers for ongoing clinical trials of metformin in this obesity-driven disease.