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Purpose To evaluate the role of substance P (SP)/neurokinin-1 receptor (NK1R) system in the regulation of pathologic corneal lymphangiogenesis in dry eye disease (DED). Methods Immunocytochemistry, angiogenesis assay, and Western blot analysis of human dermal lymphatic endothelial cells (HDLECs) were conducted to assess the involvement of SP/NK1R system in lymphangiogenesis. DED was induced in wild-type C57BL/6 J mice using controlled-environment chamber without scopolamine. Immunohistochemistry, corneal fluorescein staining, and phenol red thread test were used to evaluate the effect of SP signaling blockade in the corneal lymphangiogenesis. The expression of lymphangiogenic factors in the corneal and conjunctival tissues of DED mouse model was quantified by real-time polymerase chain reaction. Results NK1R expression and pro-lymphangiogenic property of SP/NK1R system in HDLECs were confirmed by Western blot analysis and angiogenesis assay. Blockade of SP signaling with L733,060, an antagonist of NK1R, or NK1R-targeted siRNA significantly inhibited lymphangiogenesis and expression of vascular endothelial growth factor (VEGF) receptor 3 stimulated by SP in HDLECs. NK1R antagonist also suppressed pathological corneal lymphangiogenesis and ameliorated the clinical signs of dry eye in vivo. Furthermore, NK1R antagonist effectively suppressed the lymphangiogenic factors, including VEGF-C, VEGF-D, and VEGF receptor 3 in the corneal and conjunctival tissues of DED. Conclusions SP/NK1R system promotes lymphangiogenesis in vitro and NK1R antagonism suppresses pathologic corneal lymphangiogenesis in DED in vivo.

Background Immunotherapies such as PD-L1 inhibitors have shown promising efficiency in breast cancer (BC) patients. However, treatment responses for immunotherapies are diverse since the immunogenicity of breast cancer is heterogeneous. Specific subtypes such as hormone receptor (HR)-positive, human EGF receptor 2 (HER2)-positive, and triple-negative breast cancer (TNBC) have shown heterogeneity in immunogenicity. Therefore, precisely identification of patients potentially benefit from immunotherapy is important. Previous studies have proved PD-L1 expression and tumor mutational burden (TMB) as predictive biomarkers for immunotherapy. Here, we report the PD-L1 expression and TMB status in Chinese BC patients with different subtypes. Using comprehensive molecular analysis, we also characterized the genomic features related to these biomarkers. Methods Tumor samples from 112 Chinese patients with BC were collected and subjected to next-generation sequencing (NGS) and immunohistochemical (IHC) analysis. NGS were performed in a laboratory accredited by College of American Pathologists (CAP) and certified by Clinical Laboratory Improvement Amendments (CLIA) using validated panel targeting 450 cancer genes. Genomic alterations, included including single base substitution, short and long insertions/deletion (Indel), copy number variation, gene fusion, and rearrangement, were assessed. Tumor mutational burden (TMB) was measured by an algorithm developed in-house. Tumor tissues were analyzed for PD-L1 expression by IHC with 22C3 or 28-8 antibodies, respectively. Results The 112 Chinese BC patients consisted of 87 HR+ (77.7%), 19 TNBC (17.0%) and 6 HR-/ HER2 + (5.3%), with a median age of 47.5 years old (range 24-81). Of all patients, 42.0% were positive for PD-L1 expression (CPS≥1), including 30.4% PD-L1 (1≥CPS>10) and 11.6% PD-L1 (CPS≥10). PD-L1 expression were observed in HR+ BCs (41.4%) as well as in TNBC (47.4%) and HR-/ HER2+ (33.3%) subtypes, no significant difference were observed among the three subtypes. 8.9% patients were TMB-High (≥10 muts/Mb) with median TMB of 3.4 muts/Mb (range 0-80.8). TMB was slightly correlated with PD-L1 expression (Kendall tau = 0.241; P =0.01). 17.0% of PD-L1 positive patients and 3.1% of PD-L1 negative patients were TMB-High (p=0.016). Patients with PD-L1 CPS≥10 had significantly higher median TMB (7.1 vs. 3.1 muts/Mb, p Conclusion Our data shows that the PD-L1 expression has no significant difference among HR+, TNBC and HR-/ HER2+ subtypes. These data provide the hypothesis that PD-L1 expression positive in different subtype breast cancers may benefit from immune treatment, including HER2 and Luminal subtypes. Otherwise, there was a correlation between PD-L1 expression and TMB, High TMB was also observed in PD-L1 negative patients, which may enrich the subgroup of patients who could benefit from immunotherapy. The verification of integrated predictive biomarkers for immunotherapy in BC is further needed. Citation Format: Ning Liao, Cao Li, Jundong Wu, Bo Chen, Guochun Zhang, Xueri Li, Liping Guo, Guangnan Wei, Jiali Lin, Yingzi Li, Yuchen Zhang, Hsiaopei Mok, Chongyang Ren, Yulei Wang, Kai Li, Cheukfai Li, Lingzhu Wen, Minghan Jia, Tengjiao Lin, Chunyan Cheng. Pd-l1 expression among different subtypes of chinese breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-44.

Introduction We retrospectively examined progression-free survival (PFS) and response by ALK fluorescence in situ hybridization (FISH) status in patients with advanced ALK immunohistochemistry (IHC)-positive NSCLC in the ALEX study. Methods A total of 303 treatment-naive patients were randomized to receive twice-daily alectinib 600 mg or crizotinib 250 mg. ALK status was assessed centrally using Ventana ALK (D5F3) CDx IHC and Vysis ALK Break Apart FISH Probe Kit. Primary end point is investigator-assessed PFS. Secondary end points of interest are objective response rate and duration. Results Investigator-assessed PFS was significantly prolonged with alectinib versus crizotinib in ALK IHC-positive and FISH-positive tumors (n = 203, 67%) (hazard ratio [HR] = 0.37, 95% confidence interval [CI]: 0.25–0.56; p Conclusions Outcomes of patients with ALK IHC-positive and FISH-positive and ALK IHC-positive and FISH-uninformative NSCLC were similar to those of the overall ALEX population. These results suggest that Ventana ALK IHC is a standard testing method for selecting patients for treatment with alectinib.

BackgroundPembrolizumab is a standard-of-care first-line treatment for advanced/metastatic NSCLC, either as monotherapy (for patients with PD-L1 tumor proportion score [TPS] ≥1%) or combined with platinum chemotherapy. An improved OS benefit has been demonstrated for both pembrolizumab monotherapy and pembrolizumab plus chemotherapy in patients with higher tumor PD-L1 expression, and for pembrolizumab monotherapy in patients with higher tissue tumor mutation burden (tTMB). Mutations in KRAS occur relatively frequently in patients with nonsquamous NSCLC but infrequently in those with squamous NSCLC; most mutations are in codon 12. Notably, the pembrolizumab OS treatment effect was not diminished in patients with KRAS G12C mutations in phase 3 studies evaluating pembrolizumab monotherapy and pembrolizumab in combination with chemotherapy.1 2 Herein we describe prevalence of KRAS mutations among patients with advanced nonsquamous NSCLC from two phase 3 clinical studies evaluating first-line pembrolizumab (KEYNOTE-042 and KEYNOTE-189) and the relationship of such mutations with select patient characteristics.MethodsKEYNOTE-042 (NCT02220894) evaluated pembrolizumab versus platinum-based chemotherapy for advanced PD-L1–positive NSCLC (any histology) without EGFR/ALK alterations. KEYNOTE-189 (NCT02578680) evaluated pembrolizumab plus platinum-based chemotherapy versus platinum-based chemotherapy alone for metastatic nonsquamous NSCLC without EGFR/ALK alterations irrespective of tumor PD-L1 expression. Whole-exome sequencing of tumor tissue and matched normal DNA (blood) was performed for patients with nonsquamous histology. PD-L1 TPS was evaluated using the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies, Carpinteria, CA, USA). Prevalence of KRAS mutations and their relationships with TMB, PD-L1 TPS, and smoking status were analyzed descriptively.Results590 patients with nonsquamous NSCLC were included in these analyses (KEYNOTE-042, n=301; KEYNOTE-189, n=289). Overall, 42.9% of patients had tTMB ≥175 mut/exome, 81.4% were current/former smokers and, 40.3%, 42.7%, and 16.9% had PD-L1 TPS ≥50%, 1–49% and KRAS G12C, G12D, and G12V mutations occurred in 11.0%, 4.1%, and 5.4% of patients, respectively. Prevalence of KRAS mutations by patient characteristics is summarized in the table (table 1). KRAS G12C mutations occurred almost exclusively in current/former smokers. KRAS G12C was enriched in tumors with tTMB ≥175 mut/exome and tumors with PD-L1 TPS ≥50%. Prevalence was highest in tumors with both tTMB ≥175 mut/exome and PD-L1 TPS ≥50%.Abstract 364 Table 1KRAS Mutation PrevalenceConclusionsKRAS G12C mutations occurred with moderate frequency in patients with nonsquamous NSCLC, with most occurring in current/former smokers. KRAS G12C mutations occurred at higher frequency in patient subgroups defined by higher tTMB and PD-L1 TPS.AcknowledgementsMedical writing assistance was provided by Christabel Wilson, MSc, of ICON plc (North Wales, PA, USA), funded by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.Trial RegistrationKEYNOTE-042, ClinicalTrials.gov, NCT02220894; KEYNOTE-189, ClinicalTrials.gov, NCT02578680ReferencesGadgeel S, Rodriguez-Abreu D, Felip E, et al. KRAS mutational status and efficacy in KEYNOTE-189: pembrolizumab (pembro) plus chemotherapy (chemo) vs placebo plus chemo as first-line therapy for metastatic non-squamous NSCLC. Ann Oncol 2019;30(suppl 11):xi64-xi5.Herbst RS, Lopes G, Kowalski DM, et al. Association of KRAS mutational status with response to pembrolizumab monotherapy given as first-line therapy for PD-L1-positive advanced non-squamous NSCLC in KEYNOTE-042. Ann Oncol 2019;30(suppl 11):xi63-xi4.Ethics ApprovalFor both trials, the protocol and all amendments were approved by the appropriate ethics committee at each center, the study was conducted in accordance with the standards of Good Clinical Practice. Patients provided written informed consent before enrollment.

Background Retinal degeneration is a condition ensued by various ocular disorders such as artery occlusion, diabetic retinopathy, retrolental fibroplasia and retinitis pigmentosa which cause abnormal loss of photoreceptor cells and lead to eventual vision impairment. No efficient treatment has yet been found, however, the use of stem cell therapy such as bone marrow and embryonic stem cells has opened a new treatment modality for retinal degenerative diseases. The major goal of this study is to analyze the potential of endothelial progenitor cells derived from bone marrow to differentiate into retinal neural cells for regenerative medicine purposes. Methods In this study, endothelial progenitor cells were induced in-vitro with photoreceptor growth factor (taurine) for 21 days. Subsequently, the morphology and gene expression of CRX and RHO of the photoreceptors-induced EPCs were examined through immunostaining assay. Findings The results indicated that the induced endothelial progenitor cells demonstrated positive gene expression of CRX and RHO. Our findings suggested that EPC cells may have a high advantage in cell replacement therapy for treating eye disease, in addition to other neural diseases, and may be a suitable cell source in regenerative medicine for eye disorders.

Background and study aims Endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) or biopsy (FNB) is an indispensable diagnostic tool. Improvements in needling technique have led to increasing tissue yields. Blood clogging of the needle can cause difficulties with specimen handling and stylet passage, which improves when the needle is primed with heparin before use. However, the effect of heparin on cytology, histology or immunochemistry (IHC) of FNA and FNB specimens is unknown. The goal of the study was to evaluate heparin priming on cytologic/histologic appearance, IHC staining, ease of stylet passage, and specimen bloodiness. Patients and methods This was a retrospective study of patients undergoing EUS-FNA/FNB. Needle sizes were 25 gauge (g), 22 g, and 19 g. Heparin priming of the needle was done and the stylet replaced (“dry heparin”) or suction attached without replacing the stylet (“wet heparin”). Smears and cellblocks were examined by pathologists, and IHC staining were done as needed. Specimen bloodiness was compared with matched controls. Results Adequate tissue yields were obtained in all samples (37 heparin, 36 no heparin). Heparin priming did not exhibit negative effects on cytologic or histologic interpretation of the specimens, nor IHC. There was no difference in cellblock bloodiness between the heparin primed needle specimens and the non-heparin control group. Conclusions Heparin priming of EUS-FNA or FNB needles does not negatively affect cytologic or histologic interpretation, nor interfere with IHC. In addition, heparin priming does not increase specimen bloodiness. When the “wet suction” technique is used for EUS-FNA, heparin priming can be used instead of saline priming of the EUS needle.

The extent to which intratumoral heterogeneity of programmed death ligand 1 (PD-L1) expression causes discordance of PD-L1 expression between paired samples remains unclear. Here, PD-L1 status was compared between whole sections from NSCLCs and the corresponding tissue microarrays (TMAs) serving as surrogate biopsy specimens.PD-L1 expression was evaluated by 22C3 immunohistochemistry assay on 190 archival surgical specimens and matched to the TMA results. PD-L1 expression was determined by the tumor proportion score (TPS) and classified as TPS lower than 1%, TPS of 1% to 49%, and TPS of 50% or higher. Agreement statistics were used.The percentage of PD-L1 expression on tumor cells differed greatly between individual TMAs and matched surgical specimens. When PD-L1 TPS was adopted, a total of 36 of 190 discordance cases (18.9%) were observed, with a κ-value of 0.630 between paired samples. The TMAs underestimated or overestimated PD-L1 status in 19 of 36 (52.8%) and 17 of 36 (47.2%) of the matched surgical specimens, respectively (p = 0.118). The discordance rate was much lower in cases with a PD-L1 TPS lower than 1% compared with in cases with a TPS of 1% to 49% and TPS of 50% or higher (18.4% versus 56.7% and 43.3%, p0.001). When a TPS of 50% or higher was used as the cutoff, the discordance rate of PD-L1 TPS less than 50% was further reduced to 7.5%. Such discrepancies were due mainly to intratumoral heterogeneity of PD-L1 expression and nonsignificant association with clinicopathological features.PD-L1 expression in TMAs correlates moderately well with that in the corresponding surgical specimens, indicating that evaluating PD-L1 expression in diagnostic biopsy specimens could be misleading in defining sensitivity to pembrolizumab treatment yet may be reliable as a way to exclude patients with a PD-L1 TPS less than 50% from first-line pembrolizumab treatment.

Distraction osteogenesis is widely used in many clinical situations, but distraction in cases accompanying nerve injury has been avoided due to concern of unfavorable effect on nerve regeneration by traction. This study evaluated the feasibility of early distraction lengthening after neurorrhaphy. Thirty-six rats were evenly distributed into 3 groups (12 rats in each group); neurorrhaphy and distraction (group I), neurorrhaphy and osteotomy without distraction (group II), and only distraction without neurorrhaphy (group III), respectively. After osteotomy on the right tibia, distraction started after 1 week and was continued for 40 days with 0.25 mm per day. Histological evaluation was carried out to identify nerve regeneration at 4, 8, and 12 weeks after surgery. Walking tract analysis was performed to assess the functional recovery preoperatively and 1, 4, 8, and 12 weeks postoperatively. Histologically, axon number ratio was significantly impaired in group I (0.48 ± 0.14) and group II (0.53 ± 0.13) compared with group III (0.88 ± 0.04) at 4 weeks (P = 0.020). There was no significant difference at both 8 and 12 weeks. Walking tract analysis showed significant differences between groups I and III (-40.5 ± 4.3), and groups II and III (-35.5 ± 5.0) at 1 week (P = 0.001), but no difference was observed at 8 and 12 weeks. Distraction osteogenesis in early stage after nerve repair is safe and effective, when performed at a rate of 0.25 mm per day in rats.

To investigate the prognostic and biologic significance of immune-related gene expression in high grade serous ovarian cancer (HGSOC). Gene expression dependent survival analyses for a panel of immune related genes were evaluated in HGSOC utilizing The Cancer Genome Atlas (TCGA). Prognostic value of LCK was validated using IHC in an independent set of 72 HGSOC. Prognostic performance of LCK was compared to cytolytic score (CYT) using RNAseq across multiple tumor types. Differentially expressed genes in LCK high samples and gene ontology enrichment were analyzed. High pre-treatment LCK mRNA expression was found to be a strong predictor of survival in a set of 535 ovarian cancers. Patients with high LCK mRNA expression had a longer median progression free survival (PFS) of 29.4 months compared to 16.9 months in those without LCK high expression (p = 0.003), and longer median overall survival (OS) of 95.1 months versus 44.5 months (p = 0.001), which was confirmed in an independent cohort by IHC (p = 0.04). LCK expression was compared to CYT across tumor types available in the TCGA and was a significant predictor of prognosis in HGSOC where CYT was not predictive. Unexpectedly, LCK high samples also were enriched in numerous immunoglobulin-related and other B cell transcripts. LCK is a better prognostic factor than CYT in ovarian cancer. In HGSOC, LCK high samples were characterized by higher expression of immunoglobulin and B-cell related genes suggesting that a cooperative interaction between tumor infiltrating T and B cells may correlate with better survival in this disease.

The ryanodine receptor 1 (RYR1) is a calcium release channel essential for excitation-contraction coupling in the sarcoplasmic reticulum of skeletal muscles. Dominant variants in the RYR1 have been well associated with the known pharmacogenetic ryanodinopathy and malignant hyperthermia. With the era of next-generation gene sequencing and growing number of causative variants, the spectrum of ryanodinopathies has been evolving with dominant and recessive variants presenting with RYR1-related congenital myopathies such as central core disease, minicore myopathy with external ophthalmoplegia, core-rod myopathy, and congenital neuromuscular disease. Lately, the spectrum was broadened to include fetal manifestations, causing a rare recessive and lethal form of fetal akinesia deformation sequence syndrome (FADS)/arthrogryposis multiplex congenita (AMC) and lethal multiple pterygium syndrome. Here we broaden the spectrum of clinical manifestations associated with homozygous/compound heterozygous RYR1 gene variants to include a wide range of manifestations from FADS through neonatal hypotonia to a 35-year-old male with AMC and PhD degree. We report five unrelated families in which three presented with FADS. One of these families was consanguineous and had three affected fetuses with FADS, one patient with neonatal hypotonia who is alive, and one individual with AMC who is 35 years old with normal intellectual development and uses a wheelchair. Muscle biopsies on these cases demonstrated a variety of histopathological abnormalities, which did not assist with the diagnostic process. Neither the affected living individuals nor the parents who are obligate heterozygotes had history of malignant hyperthermia.

Summary Objective To explore the effects of Mg2+ on the expression of osteoarthritic markers in human cartilage and synovium tissue explants. To investigate the therapeutic effect of intra-articular injection of Mg2+ in an established rat OA (Osteoarthritis) model of anterior cruciate ligament transection with partial medial meniscectomy (ACLT + PMM). Design Human cartilage and synovium explants were collected from total knee replacement surgeries and incubated with MgCl2 (20 mmol/L) in vitro. A rat OA model was established by ACLT + PMM surgery in 450–500 g male Sprague Dawley (SD) rats. To select the optimal dose, intra-articular injections of MgCl2 (0.05, 0.5, 5 mol/L) were performed at 4 weeks after the surgery every 3 days for 2 weeks. The effect of optimized MgCl2 was further determined by histology, immunohistochemistry, and quantitative real-time polymerase chain reaction. Results The expressions of osteoarthritic markers in human cartilage and synovium explants were inhibited by Mg2+ in vitro. Immunohistochemical analysis further suggested the inhibitory effects of Mg2+ on the expression of MMP-13 and IL-6 in the human tissue explants. Cartilage degeneration and synovitis in ACLT + PMM rats were significantly improved by intra-articular injections of Mg2+ (0.5 mol/L). Immunohistochemical analysis also showed the regulatory effects of Mg2+ on osteoarthritic markers in both cartilage and synovium in rats, consistent with in vitro results. Conclusion Intra-articular injections of Mg2+ at 0.5 mol/L attenuate the progression of OA in the ACLT + PMM rat model. Such effect was at least in part explained by the promotion of cartilage matrix synthesis and the suppression of synovial inflammation.

Aim Colon cancer is one of the leading causes of cancer-related mortality. However, specific biomarkers for its diagnosis or treatment are not established well. Methods We developed a colon-cancer specific peptide probe using phage display libraries. We validated the specificity of this probe to colon cancer cells with immunohistochemical staining and FACS analysis using one normal cell and five colon cancer cell lines. Results This peptide probe maintained binding affinity even after serum incubation. For therapeutic applications, this peptide probe was conjugated to hematoporphyrin, a photosensitizer, which showed a significantly enhanced cellular uptake and high photodynamic effect to kill tumor cells. As another application, we made a nanoparticle modified from the peptide probe. It efficiently delivered SN-38, an anticancer drug, into tumor cells, and its tumor-targeting ability was observed in vivo after intravenous injection to the same xenograft model. Conclusion The noble dodecapeptide probe can be a promising candidate for both colon tumor diagnosis and targeted drug delivery.

As human lifespans have increased, the incidence of osteoporosis has also increased. Methylsulfonylmethane (MSM) affects the process of mesenchymal stem cell (MSC) differentiation into osteoblasts via the Janus kinase 2 (Jak2)/signal transducer and activator of transcription (STAT)5b signaling pathway, and bone morphogenetic protein 2 (BMP‑2) is also known to significantly affect bone health. In addition, the phosphorylation of small mothers against decapentaplegic (Smad)1/5/8 regulates the Runt‑related transcription factor 2 (Runx2) gene, which encodes a transcription factor for osteoblast differentiation markers. In the present study, the differentiation of MSCs treated with MSM, BMP‑2, and their combination were examined. The differentiation of osteoblasts was demonstrated through observation of morphological changes and mineralization, using alizarin red and Von Kossa staining. Western blotting analysis demonstrated that the combination of MSM and BMP-2 increased the phosphorylation of the BMP signaling-associated protein, Smad1/5/8. Combination of MSM and BMP-2 significantly increased osteogenic differentiation and mineralization of the MSCs compared with either MSM or BMP-2 alone. Additionally, reverse transcription-polymerase chain reaction analysis demonstrated that combination of MSM and BMP-2 increased the expression level of the Runx2 gene and the osteoblast differentiation marker genes, alkaline phosphatase, bone sialoprotein and osteocalcin, in MSCs compared with controls. Thus, the combination of MSM and BMP-2 may promote the differentiation of MSCs into osteoblasts.

Identification of novel biomarkers and drug targets for colorectal cancer remains a critically unmet medical need. Target identification has historically been driven by preclinical models which poorly recapitulate the complex biology of human cancers. We describe a unique approach to drug target identification and comprehensive profiling directly in clinical specimens. Using the Adaptive Dynamic Artificial Polyligand Targeting (ADAPT) system, we aimed to identify such disease specific drug targets using a highly complex library of single-stranded oligodeoxynucleotides (ODNs). To generate the library, the ODNs were enriched using FFPE tissue specimens from pathologically determined colorectal cancer (CRC) and counter-selected against normal adjacent tissue (NAT) regions. Once the specificity of the library was verified via differential binding on independent CRC cases, it was used to affinity purify cognate biding partners from 393 CRC FFPE lysates with appropriate representation of cases with either KRAS, NRAS or BRAF mutations. Greater than 700 proteins were identified by mass spectrometry exclusively in the CRC FFPE tissue lysates and not in the NAT FFPE tissue lysates for all 393 CRC cases. Classification of these proteins into the druggable protein classes revealed G-protein coupled receptors, transporters, voltage-gated ion channels, proteases, and protein kinases. In addition, subsequent verification studies confirmed novel candidate cell surface protein targets appropriate for targeting by antibody-drug conjugates (ADC) or other biologic modalities. mRNA expression corresponding to the 700 potential targets was analyzed in over 1600 CRC FFPE tumor samples and >9000 other cancer types as well as in a broad panel of normal tissues by Whole Transcriptome Sequencing (WTS) and immunohistochemistry (IHC). We conclude that the unbiased ADAPT platform provides a robust and novel approach to drug target discovery with highly selective expression profiles directly from FFPE clinical specimens. Citation Format: Stephanie Williams, Chao Sima, Tassilo Hornung, Matthew Rosenow, Teresa Tinder, Varun Maher, Michelle Kassner, Gerri Ortiz, Perrin Mok, Nicholas Helle, Stephen Logie, Heather ONeill, Mark Miglarese, David Spetzler. Novel approach to colorectal cancer drug target discovery in human FFPE tissue using the Adaptive Dynamic Artificial Polyligand Targeting System [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 5165.

Rationale Epithelial-myoepithelial carcinoma is an extremely rare, malignant neoplasm that occurs most frequently in the major salivary glands and accounts for approximately 1% of all salivary gland neoplasms. Few reports have described the presence of epithelial-myoepithelial carcinoma in the sinonasal region; hence, the treatment guideline and prognosis remain unclear. Patient concerns We reported a case of a 75-year-old woman with complaint of nasal obstruction and frequent epistaxis for 3 years. During the nasal endoscopic examination, a mass in the left nasal cavity originating from the left nasal septum that caused bleeding on touch was observed. Diagnoses A diagnosis of epithelial-myoepithelial carcinoma was made based on the features of histopathology and immunohistochemistry of the surgical specimens. The patient was treated by surgical removal of the septal mass using the endonasal endoscopic approach. Outcomes In the serial follow-up paranasal sinus imaging and endoscopic inspection, evidence of recurrence was absent for 18 months after surgery. Lessons This report highlights a case of epithelial-myoepithelial carcinoma originating from a minor salivary gland in the nasal septum, one of the most unusual locations. Diagnosis of epithelial-myoepithelial carcinoma should be made based on the findings of immunohistochemistry of the operative specimen. Clinicians should consider complete surgical resection as the effective treatment of choice.

Immune checkpoint blockade targeting the PD-1/PD-L1 axis has recently demonstrated efficacy and promise in cancer treatment. Appropriate biomarker selection is therefore essential for improving treatment efficacy. However, the establishment of PD-L1 assay in pathology laboratories is complicated by the presence of multiple testing platforms using different scoring systems. Here we assessed the PD-L1 expression in 713 consecutive non-small cell lung carcinomas by four commercially available PD-L1 immunohistochemical assays, namely, 22C3, 28-8, SP142 and SP263. The analytical performances of the four assays and diagnostic performances across clinically relevant cutoffs were evaluated. The prevalence of PD-L1 (22C3) expression was 21% with a ≥50% cutoff and 56% with a ≥1% cutoff. High PD-L1 expression (using a ≥50% cutoff) was significantly associated with male sex (P = 0.001), ever smoking history (P 0.001), squamous cell carcinoma (P = 0.001), large cell carcinoma (P 0.001), lymphoepithelioma-like carcinoma (P = 0.006), sarcomatoid carcinoma (P 0.001), mutant KRAS (P = 0.005) and wild-type EGFR (P = 0.003). Elevated PD-L1 expression was also significantly associated with shorter survival in patients with adenocarcinoma (log-rank P = 0.026) and remained an independent prognostic factor by multivariable analysis. Among the four assays, 22C3, 28-8 and SP263 were highly concordant for tumor cell scoring. With a cutoff of ≥50% (i.e., the threshold for first-line patient selection), inter-rater agreement was high among the three assays with percentage agreement97%. In conclusion, three PD-L1 assays showed good analytical performance and a high agreement with each other, but not all cases were correctly classified using the same clinical cutoff. Further studies comparing the predictive value of these assays are required to address the interchangeability of these assays for clinical use.

Purpose: To investigate a sequential chronological change in tip cells during the development of the retinal vasculature in newborn mice. Materials and Methods: Newborn C57BL/6 mice were used for this study. To elucidate the patterns in the developing retinal vasculature, histology, and immunohistochemistry—antiplatelet endothelial cell adhesion molecule-1, anticollagen type IV, isolectin IB4—were performed on sections of mouse retina on postnatal days (P)-4, -8, and -12. Staining patterns of isolectin IB4-stained arterial and venous tip cells were compared in retinal wholemounts, in which the numbers and characteristics of tip cells were compared between arteries and veins on P-4, -6, and -8. In addition, vascular densities and branching patterns were compared between arterial and venous vascular forefront areas. Results: Tip cells in the superficial vascular plexus were observed until P-8. The number of tip cells was highest on P-6, decreasing dramatically from P-6 to P-8 (P-4, 165.2 ± 10.1, n = 17; P-6, 183.8 ± 19.4, n = 15; P8, 21.4 ± 6.4, n = 15) (p < 0.05, respectively, t-test). There was a greater number of tip cells in veins versus arteries on P-4 and P-6 (P-4, 91.0 ± 9.2 veins versus 74.2 ± 10.4 arteries; P-6, 104.0 ± 10.2 veins versus 79.8 ± 11.3 arteries) (p < 0.05, respectively). Arterial tip cells had thinner and longer sprouts compared with venous tip cells (basal thickness: 15.7 ± 8.7 veins versus 9.9 ± 3.5 μm arteries) (length, 20.3 ± 9.1 veins versus 37.1 ± 13.2 μm arteries on P-4) (p < 0.05, respectively). Vessel areas and densities of vascular branch points were significantly higher around veins compared to arteries (vessel areas: 58.9 ± 1.2% veins versus 40.8 ± 1.9% arteries; vascular branch points, 1371.9 ± 136.7/mm2 veins versus 1046.7 ± 175.5/mm2 arteries) (p < 0.05, respectively). Conclusion: The number of tip cells increased to a greater extent in the superficial vascular plexus of veins versus arteries until P-6. Consequently, there are more vessel areas and vascular branch points near retinal veins versus arteries. Arterial tip cells are longer and thinner than the shorter and thicker venous tip cells.

Inhibitor of apoptosis proteins (IAPs) plays an important role in controlling cancer cell survival. IAPs have therefore attracted considerable attention as potential targets in anticancer therapy. In this study, we investigated the anti-tumor effect of AZD5582, a novel small-molecule IAP inhibitor, in human pancreatic cancer cells. Treating human pancreatic cancer cells with AZD5582 differentially induced apoptosis, dependent on the expression of p-Akt and p-XIAP. Moreover, the knockdown of endogenous Akt or XIAP via RNA interference in pancreatic cancer cells, which are resistant to AZD5582, resulted in increased sensitivity to AZD5582, whereas ectopically expressing Akt or XIAP led to resistance to AZD5582. Additionally, AZD5582 targeted cIAP1 to induce TNF-α-induced apoptosis. More importantly, AZD5582 induced a decrease of Mcl-1 protein, a member of the Bcl-2 family, but not that of Bcl-2 and Bcl-xL. Interestingly, ectopically expressing XIAP and cIAP1 inhibited the AZD5582-induced decrease of Mcl-1 protein, which suggests that AZD5582 elicits Mcl-1 decrease for apoptosis induction by targeting of XIAP and cIAP1. Taken together, these results indicate that sensitivity to AZD5582 is determined by p-Akt-inducible XIAP phosphorylation and by targeting cIAP1. Furthermore, Mcl-1 in pancreatic cancer may act as a potent marker to analyze the therapeutic effects of AZD5582.

Purpose: The purpose of this study was to establish a simple, xeno-feeder-free method for cultivating human corneal epithelial cells. Methods: Limbal tissue explants from a cadaver were cultured in Iscove's modified Dulbecco's medium and low-calcium Panserin 801 medium in a 1:1 ratio. The outgrowing cells were characterized by flow cytometry, immunohistochemistry and real-time PCR (rtPCR). Limbal epithelial cells were expanded in a xeno-feeder-free, low-calcium medium and airlifted for 2 weeks each. Results: Migration of fibroblast-like stromal cells initially occurred from the limbal explants, and then epithelial cells migrated and grew on the stromal cells as an autofeeder layer. After airlifting, the cultured epithelium consisted of two to three layers. The cultured cells expressed stem cell-associated markers (ABCG2 and ΔNp63), differentiation markers (CK3 and CK12) and extracellular matrix-associated markers (lumican and decorin). rtPCR showed increased expression of markers for epithelial progenitor cells compared to fresh limbal tissue. Side population cells comprised 0.43 ± 0.04% of the cells (n = 5) in the primary culture. Flow cytometry showed that 49.12, 40.44 and 44.55% of the cells from the explants expressed E-cadherin, ΔNp63 and ABCG2, respectively. Conclusion: This explant culture system using stromal cells as an autofeeder layer was useful in expanding human corneal epithelial cells. This system may offer clinical insight for the expansion of limbal progenitor cells for the reconstruction of the ocular surface.

Background Osteosarcoma (OSA) is a common malignant bone tumor of large breed dogs that occurs at predictable anatomic sites. At the time of initial diagnosis, most affected dogs have occult pulmonary metastases. Even with aggressive surgical treatment combined with chemotherapy, the majority of dogs diagnosed with OSA live less than 1 year from the time of diagnosis. The ability to identify canine OSA cases most responsive to treatment is needed. In humans, OSA is also an aggressive tumor that is histologically and molecularly similar to canine OSA. The expression of the tumor suppressor gene product P16 by human OSA tissue has been linked to a favorable response to chemotherapy. Results We identified an antibody that binds canine P16 and developed a canine OSA tissue microarray in order to test the hypothesis that P16 expression by canine OSA tissue is predictive of clinical outcome following amputation and chemotherapy. Although statistical significance was not reached, a trend was identified between the lack of canine OSA P16 expression and a shorter disease free interval. Conclusions The identification of a molecular marker for canine OSA is an important goal and the results reported here justify a larger study.

Introduction The development of reliable gene expression profiling technology increasingly impacts our understanding of lung cancer biology. Here, we used RNA sequencing (RNA-Seq) to compare the transcriptomes of non-small cell lung cancer (NSCLC) and normal lung tissues and to investigate expression in lung cancer tissues. Methods We enrolled 88 male patients (mean age, 61.2 years) with NSCLC. RNA-Seq was performed on 88 pairs of NSCLC tumor tissue and non-tumor tissue from 54 patients with adenocarcinoma and 34 patients with squamous cell carcinoma. Immunohistochemistry was performed to validate differential candidate gene expression in a different NSCLC group. Results RNA-Seq produced 25.41×10 6 (±8.90×10 6 ) reads in NSCLC tissues and 24.70×10 6 (±4.70×10 6 ) reads in normal lung tissues [mean (±standard deviation)]. Among the genes expressed in both tissues, 335 were upregulated and 728 were downregulated ≥2-fold ( p CBX3 , GJB2 , CRABP2 , and DSP – not previously reported in lung cancer were studied further. Their altered expression was verified by immunohistochemistry in a different set of NSCLC tissues ( n =154). CBX3 was positive in 90.3% (139 cases) of the samples; GJB2 , in 22.7% (35 cases); CRABP2 , in 72.1% (111 cases); and DSP , in 17.5% (27 cases). The positive rate of CRABP2 was higher in adenocarcinoma than squamous cell carcinoma ( p Conclusions CBX3 and CRABP2 expression was markedly increased in lung cancer tissues and especially CRABP2 may be promising candidate genes in lung adenocarcinoma.

Supplemental Digital Content is available in the text, Programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) assays are widely used for complementary or companion diagnostic purposes during treatment with immune checkpoint inhibitors. However, limited information is available on the clinical reliability of the PD-L1 IHC assay using small biopsy samples. Participants included 46 patients with nonsmall cell lung cancer who underwent PD-L1 testing using 3 PD-L1 IHC assays (22C3, SP142, and SP263) for both small biopsy samples and surgical specimens from November 2017 to June 2018. The PD-L1 IHC assay results were analyzed with cut-off values of 1%, 5%, 10%, and 50%. The PD-L1 IHC results obtained from the surgical specimens were regarded as the reference values. The 22C3, SP142, and SP263 PD-L1 IHC assays were performed in 26 (57%), 20 (43%), and 46 (100%) patients, respectively. Biopsy methods included radial probe endobronchial ultrasound using a guide sheath, endobronchial ultrasound-guided transbronchial needle aspiration, bronchoscopic biopsy, and percutaneous needle aspiration in 26 (57%), 4 (9%), 12 (25%), and 4 (9%) patients, respectively. The 22C3, SP142, and SP263 PD-L1 assays had concordance rates of 73–96, 65–80, and 72%–91%, respectively, compared with the reference values. PD-L1 testing with 3 commercial PD-L1 IHC assays using small biopsy samples is reliable in patients with nonsmall cell lung cancer.

Summary Background Since March, 2013, an avian-origin influenza A H7N9 virus has caused severe pneumonia in China. The aim of this study was to investigate the pathogenesis of this new virus in human beings. Methods We obtained ex-vivo cultures of the human bronchus, lung, nasopharynx, and tonsil and in-vitro cultures of primary human alveolar epithelial cells and peripheral blood monocyte-derived macrophages. We compared virus tropism and induction of proinflammatory cytokine responses of two human influenza A H7N9 virus isolates, A/Shanghai/1/2013 and A/Shanghai/2/2013; a highly pathogenic avian influenza H5N1 virus; the highly pathogenic avian influenza H7N7 virus that infected human beings in the Netherlands in 2003; the 2009 pandemic influenza H1N1 virus, and a low pathogenic duck H7N9 virus that was genetically different to the human disease causing A H7N9 viruses. Findings Both human H7N9 viruses replicated efficiently in human bronchus and lung ex-vivo cultures, whereas duck/H7N9 virus failed to replicate in either. Both human A H7N9 viruses infected both ciliated and non-ciliated human bronchial epithelial cells and replicated to higher titres than did H5N1 (p

Introduction The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) fusion gene has been identified as a potent oncogenic driver in non–small-cell lung cancer, in particular adenocarcinoma (ADC). It defines a unique subgroup of lung ADC, which may be responsive to ALK inhibitors. Detection of ALK rearrangement by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR) is considered to be the standard procedure, but each with its own limitation. We evaluated the practical usefulness of immunohistochemistry (IHC) to detect ALK expression as a reliable detection method of ALK rearrangement in lung ADC. Methods We tested 373 lung ADCs for ALK rearrangement by IHC and FISH. Multiplex RT-PCR was performed to confirm the fusion variants. Results Twenty-two of 373 lung ACs (5.9%) were positive for ALK immunoreactivity. ALK-positive tumor cells demonstrated strong and diffused granular staining in the cytoplasm. All the ALK IHC-positive cases were confirmed to harbor ALK rearrangement, either by FISH, or RT-PCR. Two cases with positive ALK protein expression, but negative for breakapart FISH signal were shown to harbor EML4-ALK variant 1 by RT-PCR. None of the ALK IHC-negative cases were FISH-positive. In addition, we identified a novel EML4-ALK fusion variant (E3:ins53A20), and its potent transformation potential has been confirmed by in vivo tumorigenicity assay. Conclusion IHC can effectively detect ALK rearrangement in lung cancer. It might provide a reliable and cost-effective diagnostic approach in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy.

Background: Microsatellite instability (MSI) is a form of genetic instability characterized by new alleles not present in the normal genotype. This mutation occurs by altered DNA mismatch repair (MMR) genes. Studies of limited numbers of patients have reported conflicting results regarding the association of the MSI phenotype with gastric cancer. This study aims to evaluate the clinical significance of mismatch repair genes in gastric cancer. Materials and Methods: We studied 156 gastric cancer patients who underwent gastrectomy from March 2010 to February 2011 in our hospital. Mismatch repair status was determined by the immunohistochemical analysis of human MutL Homolog 1 (hMLH1) and human MutS Homolog 2 (hMSH2) expression. Results: Seventeen (10.9%) cases did not express hMLH1 but all cases expressed hMSH2. In univariate analyses, the expression of hMLH1 was associated with age, nodal status, and Lauren's classification. In multivariate analyses, there was no statistically significant association between the loss of hMLH1 expression and selected clinical parameters. Conclusion: The expression of hMLH1 was associated with age, nodal status, and Lauren's classification. Our results suggest that MMR gene abnormalities play an important role in the tumorigenesis of patients demonstrating gastric cancer.

Purpose: Among cell adhesion molecules, serum levels of intercellular adhesion molecule-1 and E-selectin are known to be correlated with the metastatic potential of gastric cancer. In the present study, the authors investigated the expression of intercellular adhesion molecule-1 and E-selectin in gastric cancer tissues and cultured gastric cancer cells, and examined their clinical value in gastric cancer. Materials and Methods: The protein was extracted from gastric cancer tissues and cultured gastric cancer cells (MKN-28 and Kato-III) and the expression of intercellular adhesion molecule-1 and E-selectin was examined by western blotting. The clinical significance of intercellular adhesion molecule-1 and E-selectin was explored, using immunohistochemical staining of specimens from 157 gastric cancer patients. Results: In western blot analysis, the expressions of intercellular adhesion molecule-1 in gastric cancer tissues and cultured gastric cancer cells were increased, however, E-selectin in gastric cancer tissues and cells were not increased. Among 157 gastric cancer patients, 79 patients (50%) were intercellular adhesion molecule-1 positive and had larger tumor size, an increased depth of tumor invasion, lymph node metastasis and perineural invasion. The intercellular adhesion molecule-1 positive group showed a higher incidence of tumor recurrence (40.5%), and a poorer 3-year survival than the negative group (54.9 vs. 85.9%, respectively). Conclusions: Intercellular adhesion molecule-1 is overexpressed in gastric cancer tissues and cultured gastric cancer cells, whereas Eselectin is not overexpressed. Increased expression of intercellular adhesion molecule-1 in gastric cancer could be related to the aggressive nature of the tumor, and has a poor prognostic effect on gastric cancer.

Carcinosarcoma of the salivary glands is a rare neoplasm, and the minimally invasive form constitutes a subgroup with a more favorable prognosis. The cytomorphologic features of this neoplasm can be appreciated on fine-needle aspiration biopsy.We present a patient with a minimally invasive carcinosarcoma ex non-recurrent pleomorphic adenoma (Ca ex PA) who underwent initial fine-needle aspiration biopsy followed by surgical resection. The tumor was composed predominantly of a light microscopic pleomorphic high-grade sarcoma exhibiting partial myoepithelial immunohistochemical features, with a minor component of in situ and invasive salivary duct carcinoma (10%). A limited area with features of a hyalinized pleomorphic adenoma was identified.This is the third case report of the cytological features of Ca ex PA of the salivary gland, with histologic correlation. It further illustrates the oncogenic relationship between epithelial and myoepithelial elements in the early stages of carcinosarcomatous transformation. © 2016 Wiley Periodicals, Inc. Head Neck 38: E2483-E2489, 2016.

Mesenchymal stromal cells (MSC) are an attractive cell-targeting vehicle for gene delivery. MIDGE (an acronym for Minimalistic, Immunologically Defined Gene Expression) construct is relatively safer than the viral or plasmid expression system as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. The objective of this study was to test the ability of the human MSC (hMSC) to deliver the erythropoietin (EPO) gene in a nude mice model following nucleofection using a MIDGE construct. hMSC nucleofected with MIDGE encoding the EPO gene was injected subcutaneously in Matrigel at the dorsal flank of nude mice. Subcutaneous implantation of nucleofected hMSC resulted in increased hemoglobin level with presence of human EPO in the peripheral blood of the injected nude mice in the first two weeks post-implantation compared with the control groups. The basal layer of the hair shaft in the dermal layer was found to be significantly positive for immunohistochemical staining of a human EPO antibody. However, only a few basal layers of the hair shaft were found to be positively stained for CD105. In conclusion, hMSC harboring MIDGE-EPO could deliver and transiently express the EPO gene in the nude mice model. These cells could be localized to the hair follicle and secreted EPO protein might have possible role in hair regeneration.

Backgrounds and Objectives The male predominance of gastric cancer suggests that female sex hormones may have a protective effect against gastric cancer. We evaluated the expression of estrogen receptors in gastric cancer tissue and cells and the clinical significance of ER-β expression in gastric cancer. Method ER-α, ER-β proteins extracted from normal stomach, gastric cancer tissues, and cultured gastric cancer cells (KATO-III, mkn28, mkn45, and mkn74) were assessed by Western blot analysis. The clinical significance of ER-β was explored using tissue microarray methods and immunohistochemical staining of specimens from 148 gastric cancers. Results Both ER-α and β protein expression were noted in normal and gastric cancer tissues. However, in cultured gastric cells, only ER-β was noted in mkn28 and mkn74. Of 148 gastric cancers, 67 (45.3%) were ER-β positive. The ER-β positive group was associated with lower tumor stage, Lauren's intestinal type, negative perineural invasion, and free of recurrence. The ER-β positive group had a better 3-year survival compared with the negative group in survival analysis. Conclusion Our results suggest that the presence of ER-β in gastric cancer could have a protective effect against invasiveness of gastric cancer. Further studies are needed to clarify the role of ER-β in gastric cancers. J. Surg. Oncol. 2012; 106:456–461. © 2012 Wiley Periodicals, Inc.

Summary Background: To identify meningioma-specific proteins, cerebrospinal fluid (CSF) from 4 patients with a menin -gioma and 4 patients with a non-brain tumorous lesion were analyzed. Material/Methods: Two-dimensional electrophoresis and electrospray quadrupole time-of-flight tandem mass spec-trometry analyses revealed 10 unique spots, containing 11 independent proteins (spot #2 and #4 each contained 2 proteins and spot #3 was not identified) were evident in CSF associated with hu -man meningioma: serum albumin precursor (3 different isoforms), Apolipoprotein E (Apo E), Apolipoprotein J precursor (Apo J), Transthyretin precursor (TTR), Prostaglandin D2 synthase 21kDa (PTGDS), proapolipoprotein, Chain D hemoglobin Ypsilanti, alpha-1-antitrypsin (AAT), and beta-2-microglobulin precursor (b2M). Results: The contents of Apo E, Apo J and AAT were increased, while PTGDS, TTR and b2M were decreased. Conclusions: The results observed by 2-dimensional electrophoresis were verified by Western blot analysis. The unique proteins may represent possible candidate biomarkers of meningioma.

Purpose The results of the Iressa Pan-Asia Study (IPASS), which compared gefitinib and carboplatin/paclitaxel in previously untreated never-smokers and light ex-smokers with advanced pulmonary adenocarcinoma were published previously. This report presents overall survival (OS) and efficacy according to epidermal growth factor receptor (EGFR) biomarker status. Patients and Methods In all, 1,217 patients were randomly assigned. Biomarkers analyzed were EGFR mutation (amplification mutation refractory system; 437 patients evaluable), EGFR gene copy number (fluorescent in situ hybridization; 406 patients evaluable), and EGFR protein expression (immunohistochemistry; 365 patients evaluable). OS analysis was performed at 78% maturity. A Cox proportional hazards model was used to assess biomarker status by randomly assigned treatment interactions for progression-free survival (PFS) and OS. Results OS (954 deaths) was similar for gefitinib and carboplatin/paclitaxel with no significant difference between treatments overall (hazard ratio [HR], 0.90; 95% CI, 0.79 to 1.02; P = .109) or in EGFR mutation–positive (HR, 1.00; 95% CI, 0.76 to 1.33; P = .990) or EGFR mutation–negative (HR, 1.18; 95% CI, 0.86 to 1.63; P = .309; treatment by EGFR mutation interaction P = .480) subgroups. A high proportion (64.3%) of EGFR mutation–positive patients randomly assigned to carboplatin/paclitaxel received subsequent EGFR tyrosine kinase inhibitors. PFS was significantly longer with gefitinib for patients whose tumors had both high EGFR gene copy number and EGFR mutation (HR, 0.48; 95% CI, 0.34 to 0.67) but significantly shorter when high EGFR gene copy number was not accompanied by EGFR mutation (HR, 3.85; 95% CI, 2.09 to 7.09). Conclusion EGFR mutations are the strongest predictive biomarker for PFS and tumor response to first-line gefitinib versus carboplatin/paclitaxel. The predictive value of EGFR gene copy number was driven by coexisting EGFR mutation (post hoc analysis). Treatment-related differences observed for PFS in the EGFR mutation–positive subgroup were not apparent for OS. OS results were likely confounded by the high proportion of patients crossing over to the alternative treatment.

Low-grade serous ovarian carcinoma is believed to arise from serous borderline ovarian tumors, yet the progression from serous borderline tumors to low-grade serous ovarian carcinoma remains poorly understood. The purpose of this study was to identify differentially expressed genes between the two groups. Expression profiles were generated from 6 human ovarian surface epithelia (HOSE), 8 serous borderline ovarian tumors (SBOT), 13 low-grade serous ovarian carcinomas (LG), and 24 high-grade serous ovarian carcinomas (HG). The anterior gradient homolog 3 (AGR3) gene was found to be highly upregulated in serous borderline ovarian tumors; this finding was validated by real-time quantitative RT-PCR, Western blotting, and immunohistochemistry. Anti-AGR3 immunohistochemistry was performed on an additional 56 LG and 103 HG tissues and the results were correlated with clinical data. Expression profiling determined that 1254 genes were differentially expressed (P < 0.005) between SBOT, LG and HG tumors. Serous borderline ovarian tumors exhibited robust positive staining for AGR3, with a lower percentage of tumor cells stained in LG and HG. Immunofluorescence staining indicated that AGR3 expression was limited to ciliated cells. Tumor samples with a high percentage (>10%) of AGR3 positively stained tumor cells were associated with improved longer median survival in both the LG (P = 0.013) and HG (P = 0.008) serous ovarian carcinoma groups. The progression of serous borderline ovarian tumors to low-grade serous ovarian carcinoma may involve the de-differentiation of ciliated cells. AGR3 could serve as a prognostic marker for survival in patients with low-grade and high-grade serous ovarian carcinomas.

Activity-dependent neuroprotective protein (ADNP) was shown to be essential for embryogenesis and brain development while NAP, an active motif of ADNP, is neuroprotective in a broad range of neurodegenerative disorders. In the present study, we examined the protective potential of ADNP/NAP in a mouse model of excitotoxic brain lesion mimicking brain damage associated with cerebral palsy. We demonstrated that NAP had a potent neuroprotective effect against ibotenate-induced excitotoxic damage in the cortical plate and the white matter of P5 mice, and moderate against brain lesions of P0 mice. In contrast, endogenous ADNP appears not to be involved in the response to excitotoxic challenge in the studied model. Our findings further show that NAP reduced the number of apoptotic neurons through activation of PI-3K/Akt pathway in the cortical plate or both PI-3K/Akt and MAPK/MEK1 kinases in the white matter. In addition, NAP prevented ibotenate-induced loss of pre-oligodendrocytes without affecting the number of astrocytes or activated microglia around the site of injection. These findings indicate that protective actions of NAP are mediated by triggering transduction pathways that are crucial for neuronal and oligodendroglial survival, thus, NAP might be a promising therapeutic agent for treating developing brain damage.

The purpose of this study is to examine the role of versican (VCAN) in advanced stage serous ovarian cancer by investigating its expression, its function, and its correlation with clinical outcomes.Microarray analysis was performed on RNA isolated from tumor and stromal components of advanced stage serous ovarian cancer and normal ovarian epithelial tissue to identify genes up-regulated in ovarian tumor stroma. Validation studies using immunohistochemistry and quantitative real-time PCR (Q-RT-PCR) was performed on one of the up-regulated genes, VCAN. Immunolocalization of VCAN (n=111) and CD31 (n=56) was done on serous ovarian tumors. CD31 staining was performed to examine microvessel density (MVD). Q-RT-PCR was performed on 65 samples to evaluate the differential expression of VCAN isoforms. Cell proliferation and invasion assays were performed to examine how V1-treated ovarian cancer cell lines and an endothelial cell line would differ from controls. Univariate survival analyses were done with VCAN expression. Correlation analysis was done with CD31, platinum resistance, and clinical data.Validation studies using Q-RT-PCR and immunohistochemistry showed significantly higher VCAN V1 isoform expression in ovarian cancer stroma compared with normal ovarian stroma and ovarian cancer cells. Correlation studies showed stromal VCAN expression was associated with poorer overall and progression-free survival, platinum resistance, and increased MVD. VCAN-treated ovarian cancer and endothelial cells showed increased invasion potential.VCAN overexpression is associated with increased MVD and invasion potential, which may lead to poorer overall and progression-free survival and platinum resistance.

Purpose In the phase III INTEREST trial, 1,466 pretreated patients with advanced non–small cell lung cancer (NSCLC) were randomly assigned to receive gefitinib or docetaxel. As a preplanned analysis, we prospectively analyzed available tumor biopsies to investigate the relationship between biomarkers and clinical outcomes. Methods Biomarkers included epidermal growth factor receptor (EGFR) copy number by fluorescent in situ hybridization (374 assessable samples), EGFR protein expression by immunohistochemistry (n = 380), and EGFR (n = 297) and KRAS (n = 275) mutations. Results For all biomarker subgroups analyzed, survival was similar for gefitinib and docetaxel, with no statistically significant differences between treatments and no significant treatment by biomarker status interaction tests. EGFR mutation–positive patients had longer progression-free survival (PFS; hazard ratio [HR], 0.16; 95% CI, 0.05 to 0.49; P = .001) and higher objective response rate (ORR; 42.1% v 21.1%; P = .04), and patients with high EGFR copy number had higher ORR (13.0% v 7.4%; P = .04) with gefitinib versus docetaxel. Conclusion These biomarkers do not appear to be predictive factors for differential survival between gefitinib and docetaxel in this setting of previously treated patients; however, subsequent treatments may have influenced the survival results. For secondary end points of PFS and ORR, some advantages for gefitinib over docetaxel were seen in EGFR mutation–positive and high EGFR copy number patients. There was no statistically significant difference between gefitinib and docetaxel in biomarker-negative patients. This suggests gefitinib can provide similar overall survival to docetaxel in patients across a broad range of clinical subgroups and that EGFR biomarkers such as mutation status may additionally identify which patients are likely to gain greatest PFS and ORR benefit from gefitinib.

Crude water extracts of 13 traditional Korean medicinal ingredients used for leiomyomal treatment were prepared and used to treat human uterine normal myometrial and leiomyomal cell cultures. All the ingredients inhibited proliferation and altered the morphology of both myometrial and leiomyomal cells. Among the 13 ingredients, n-hexane-, chloroform-, and ethylacetate-soluble fractions were extracted from seven ingredients that potently inhibited cell proliferation in their water extract form. Among these, the ethylacetate-fraction of Phlomis umbrosa and Spatholobus suberectus, and the chloroform-fraction of Curcuma zedoaria and S. suberectus inhibited leiomyomal cell proliferation significantly compared to myometrial cell proliferation. Similarly, immunohistochemical analysis showed the inhibition of transforming growth factor-beta receptor 2 in leiomyomal tissue after treatment with the fractions of the ingredients. Moreover, the chloroform-fraction of C. zedoaria was subfractionated by open column chromatography. Two of the eight subfractions (fractions 6 and 7) potently inhibited cell proliferation in leiomyoma compared to myometrium. Further study will be performed with the goal of isolating specific compounds from two effective subfractions of C. zedoaria, ethylacetate-fraction of P. umbrosa, and the ethylacetate and chloroform-fractions of S. suberectus. The present study may be helpful in developing an alternative remedy to leiomyoma with minimal side-effects compared to the current treatments.

Ovarian tumors of low-malignant potential and low-grade ovarian serous carcinomas are thought to represent different stages on a tumorigenic continuum and to develop along pathways distinct from high-grade ovarian serous carcinoma. We performed gene expression profiling on 3 normal human ovarian surface epithelia samples, and 10 low-grade and 10 high-grade ovarian serous carcinomas. Analysis of gene expression profiles of these samples has identified 80 genes up-regulated and 232 genes down-regulated in low-grade ovarian serous carcinomas. PAX2 was found to be one of the most up-regulated genes in low-grade ovarian serous carcinoma. The up-regulation of PAX2 was validated by real-time quantitative RT-PCR, Western blot and immunohistochemical analyses. Real-time RT-PCR demonstrated a statistically significant difference in PAX2 mRNA expression (expressed as fold change in comparison to normal human ovarian surface epithelia) among ovarian tumors of low-malignant potential (1837.38, N=8), low-grade (183.12, N=17), and high-grade (3.72, N=23) carcinoma samples (p=0.015). Western blot analysis revealed strong PAX2 expression in ovarian tumors of low-malignant potential (67%, N=3) and low-grade carcinoma samples (50%, N=10) but no PAX2 protein expression in high-grade carcinomas (0%, N=10). Using immunohistochemistry, tumors of low-malignant potential (59%, N=17) and low-grade carcinoma (63%, N=16) samples expressed significantly stronger nuclear staining than high-grade ovarian carcinoma samples (9.1%, N=263). Furthermore, consistent with previous immunohistochemical findings, PAX2 expression was found to be expressed in the epithelial cells of fallopian tubes but not in normal ovarian surface epithelial cells. Our findings further support the two-tiered hypothesis that tumors of low-malignant potential and low-grade ovarian serous carcinoma are on a continuum and are distinct from high-grade ovarian carcinomas. Additionally, the absence of PAX2 expression in normal ovarian epithelia but expression in fallopian tube fimbria and ciliated epithelial inclusions would suggest the potential development of tumors of low-malignant potential and of low-grade ovarian serous carcinomas from secondary Müllerian structures.

RUNX family genes, including RUNX3, are developmental regulators that are important in human cancers. The purpose of this study was to evaluate expression and oncogenic potential of RUNX3 in ovarian carcinoma.Immunohistochemical staining was performed on 60 malignant, 14 borderline, and 5 normal ovarian specimens. Correlation between RUNX3 expression with tumor histology was performed. RUNX3 expression was evaluated by quantitative real-time polymerase chain reaction (QRT-PCR) in microdissected normal and malignant epithelial ovarian tissues. Cell proliferation and viability studies were performed on cells expressing RUNX3 by lentiviral infection and cells with silenced RUNX3 expression by siRNA.RUNX3 expression by immunohistochemistry was higher in serous ovarian carcinomas versus normal ovarian epithelium (P0.001). Immunofluorescent staining confirmed upregulation of cytoplasmic RUNX3 in ovarian cancer cell lines and tissues. QRT-PCR showed higher RUNX3 mRNA expression in microdissected borderline and malignant ovarian tumor tissues compared with the normal ovarian surface epithelial cells (HOSE) (P=0.006 and P=0.023). Forced RUNX3 expression by lentiviral gene delivery in ovarian cancer cells, SKOV3, that initially showed undetectable RUNX3 expression, resulted in increased cell viability (P=0.043). Silencing RUNX3 expression by siRNA transfection into ovarian cancer cells, OVCAR429, initially expressing high levels of endogenous RUNX3 resulted in a decrease in proliferation (P=0.021).These results suggest that RUNX3 has a role in cell proliferation and viability in ovarian cancer.