IHC Studies Research Articles

Jobelyn suppresses hippocampal neuronal apoptosis and necrosis in experimental alcohol-induced brain stress

Neurodegeneration in the hippocampus is a consequence of alcohol abuse which compromises the survival of the CNS tissue and its self-renewal capacity. So far, conventional drugs have not been clinically satisfactory in ameliorating neurodegeneration, therefore there has been a surge towards exploring the potentials of nutraceuticals since they mediate their action in a multi-mechanism fashion and may have high therapeutic potentials in CNS diseases. This study, therefore, evaluated the effect of Jobelyn® supplementation in ethanol-induced neurodegeneration of the hippocampus. Adult male rats received a regimen of ethanol or ethanol plus Jobelyn®, three times daily over four days. Histological results show that Jobelyn® supplementation significantly lowered neurodegeneration in the dentate gyrus, CA1 and CA3 areas. Furthermore, IHC studies show that Jobelyn® triggered an increased expression of p53 proteins in neurons of areas CA1 and CA3, with a reduction of p53 expression in the DG and also caused reduced expression of ɤ-enolase protein in these regions. These findings suggest that Jobelyn® may be able to help to maintain neuronal survival via control of tumor antigen p53 and ɤ-enolase regulated apoptotic and necrotic processes.

Epigallocatechin-3-gallate prevents Non-alcoholic fatty liver disease by modulating liver function, lipid profiles and M1/M2 macrophage polarization

Abstract Background Here, we examine the therapeutic efficacy of Epigallocatechin-3-gallate, the most potent catechin in green tea, on nonalcoholic fatty liver disease (NAFLD), a growing epidemic in humans. Methods NAFLD was induced by high-fat diet (HFD) with 30% fructose in drinking water for 8 weeks. Mice were divided into 3 groups: model group (MD; no EGCG), low dose EGCG group (LE, 20 mg/kg/d) and high dose EGCG group (HE, 50 mg/kg/d). Liver histology, serum lipid prolife, liver function as well as liver M1/M2 polarization were evaluated. Results At the end of the experiment, the mean body weight of LE and HE mice were 39.7g and 45.7g, while it was 51.9g in the MD group (P< 0.05). Similarly, liver weights in the LE and HE groups (means = 1.44g, and 1.68g, respectively) were lower than that in MD (2.94g, P < 0.01). Liver dysfunction observed in the MD group (mean AST=133.2 U/L and ALT=183.4 U/L), was significantly improved in both LE (44.8 U/L, 86.8 U/L, respectively, P < 0.001) and HE groups (83.63 U/L, 117.6 U/L, respectively P < 0.05). Serum cholesterol, HDL, triglyceride levels were increased in the MD group, but lowered in both LE and HE mice (p<0.01). Hepatic FACS, RT-PCR and IHC studies demonstrated that the HE group had lower F4/80+ CD86+ (0.3 ×103 vs 1.5 × 104), F4/80+ MHC II+ (0.7 ×104 vs 1.3 × 104), and higher F4/80+ CD206+(2.96 ×104 vs 0.3 × 104), F4/80+ CD23+ (1.27 ×104 vs 0.65 × 104) hepatic infiltration, accompanied by down-regulated CD68 and up-regulated CD163 mRNA expression. Conclusion EGCG subdued NAFLD despite consumption of HDF and high fructose water, marked by improved liver function and serum lipid profile, as well as M1/M2 polarization. EGCG should be investigated as a therapeutic modality in human NAFLD.

ACCELERATION OF THE FIXATION OF SURGICAL AND BIOPSY MATERIAL

Objective: to develop a method for accelerating the fixation of biopsy material by adding dimexide to formalin. Material and methods. The method of adding food coloring E124 (ponso 4R) (1 mg/ml) to buffered formalin was used to study its penetration into biopsy material by leiomyomas delivered for an urgent histological study. In the test group, dimexide was added to the structure of the biopsy material as a dye penetration accelerator at a final concentration of 20%. The material was evaluated morphometrically in percents of the area of the stained areas. Results. The complete staining of all the samples of the experimental series with the addition of dimexide was observed after 12 hours of the fixation, and in the control sample only in three pieces out of six. In the remaining three samples, 75; 76 and 86% were painted. In two observations of breast cancer the expression of the fixation with the addition of 20% dimexide concentration to formalin after 12 hours corresponded to 24 hours of the fixation in the usual way. Conclusion. The addition of dimexide to buffered formalin at a final concentration of 20% made it possible to substantially accelerate the fixation without distorting the results of IGH examining the expression of markers included in the standard panel of IHC studies (Er, Pg, Ki-67, HER2).

Assessment of folate receptor alpha and beta expression in selection of lung and pancreatic cancer patients for receptor targeted therapies.

A number of folate receptor (FR) targeted small molecular drugs and monoclonal antibodies have been introduced into clinical trials to treat FR positive cancers. Because the therapeutic efficacy of these drugs depends prominently on the level of FR-α expression on the cancer cells, patients have been commonly selected for FR-targeted therapies based on the intensity of a folate-targeted radioimaging agent. Unfortunately, uptake of such imaging agents can be mediated by both major isoforms of the folate receptor, FR-α and FR-β. Logically, if the FR positive cell population in a tumor mass is dominated by FR-β positive macrophages, patients could be selected for therapy that have few FR-expressing cancer cells. Although several IHC studies have examined expression of either FR-α or FR-β, no study to date has investigated expression of both FR-α and FR-β in the same tumor mass. Herein, we utilize monoclonal antibodies specific for FR-α (mAb343) and FR-β (m909) to query each isoform's expression in a range of cancers. We show that lung and pancreatic adenocarcinomas express the full spectrum of FR-α and FR-β combinations with ~76% of lung adenocarcinomas expressing both FR-α and FR-β while pancreatic cancers express primarily FR-β. Thus, while folate-targeted imaging of lung cancer patients might accurately reflect the expression of FR-α on lung cancer cells, imaging of pancreatic cancer patients could mislead a physician into treating a nonresponding patient. Overall, these data suggest that an independent analysis of both FR-α and FR-β should be obtained to predict the potential efficacy of a folate-targeted drug.

Could the PD-1 Pathway Be a Potential Target for Treating Small Intestinal Adenocarcinoma?

The programmed death 1 (PD-1) pathway is upregulated in the immune microenvironment of many cancers. In this study, we examined the PD-1 pathway and the immune microenvironment of small intestinal adenocarcinomas by immunohistochemistry. From our department pathology archives we identified 42 small intestinal adenocarcinomas from between 2000 and 2015, with blocks available for IHC studies. Tumors were immunohistochemically stained for CD3, CD4, CD8, CD20, PD-1, and programmed death ligand 1 (PD-L1) expression. PD-1 was expressed by intratumoral and peritumoral lymphocytes in 35 of 42 (83%) cases. PD-L1 expression on tumor cells and immune cells was observed in seven of 42 (17%), and 18 of 42 (43%) cases, respectively. PD-L1 was mainly expressed by histiocytes capping cancerous glands/nests at the invasive front or by most tumor cells in medullary carcinomas. All the PD-L1+ tumors also expressed PD-1. The tumors expressing PD-L1 contained more CD3+, CD4+, and CD8+ T cells, but showed a lower CD4+/CD8+ ratio than those without expression of PD-L1. PD-1 and PD-L1 are highly expressed by most small intestinal adenocarcinomas. Blockage of the PD-1 pathway should be evaluated in the treatment of small intestinal adenocarcinomas.

Ablative therapy to induce local and systemic immune changes in patients with metastatic renal cell carcinoma.

505 Background: Ablative therapy including cryoablation or radiofrequency ablation is a therapeutic option for small renal cell carcinomas (RCC) and provides effective palliation for symptomatic metastatic lesions, with reports of associated abscopal phenomenon in small numbers of patients. Pre-clinical animal modeling has suggested potential synergy with the use of ablative therapy and immune checkpoint blockade with ipilimumab. Methods: We performed retrospective analysis of 17 patients who underwent an ablative procedure followed later by nephrectomy. These samples were analyzed by IHC and gene expression studies. We also prospectively evaluated pre and post treatment blood samples from a different cohort of 13 patients. These samples were evaluated for immune cell subsets and cytokine profiles. Results: IHC studies revealed an increased frequency of PD-1 + immune cells in post-ablative tumor samples as compared to control samples. Gene expression studies also revealed higher expression of immune gene signatures as compared to control samples. Blood samples detected changes in regulatory T cell subsets and interleukin-6 cytokine levels in the short term, which normalized by 1 month post-ablation, suggested an active downregulation of the initial immune response. Based on these data, we initiated a clinical pilot study to treat patients with anti-CTLA-4 (tremelimumab) with or without cryoablation in patients with metastatic RCC. The primary endpoint of the trial will be safety. Secondary endpoints include objective response rate, progression free survival, and immune monitoring. A total of 30 patients, 15 in each arm, will be accrued. Initial data from the clinical trial will be presented. Conclusions: Ablative therapy causes immune changes that can be detected both at the tissue and systemic level. We hope to capitalize on these changes by combining immune checkpoint blockade and ablation in an ongoing prospective clinical trial.

Assessment of BRAF V600E (VE1) protein expression and BRAF gene mutation status in codon 600 in benign and malignant salivary gland neoplasms.

The role of BRAF mutations in cancerogenesis has been demonstrated in several solid tumor types. However, in salivary gland tumors, this genetic alteration is very uncommon, and its role still remains unclear. Thus, the aim of this study was to analyze BRAF V600E (VE1) protein expression with BRAF mutation status in codon 600, in malignant and benign salivary gland tumors. Studies were performed on archived formalin-fixed paraffin-embedded tissue sections derived from 95 patients who underwent surgery for tumors of the salivary gland. Immunohistochemical staining (IHC) on tissue microarray slides was performed for evaluation of BRAF V600E (VE1) protein expression, and the automatic molecular diagnostics platform was used for the evaluation of mutations in codon 600 of BRAF gene. IHC cytoplasmic expression of BRAF V600E (VE1) protein was found in two of 95 cases: one case of adenocarcinoma NOS (one of three; 33%) and one case of carcinoma ex pleomorphic adenoma (one of five; 20%). Although, in IHC studies, nuclear BRAF V600E (VE1) protein expression was found in 14 (15%) of the analyzed cases: nine of 28 (32%) cases of pleomorphic adenoma, three of five (60%) cases of ductal carcinoma, one of nine (11%) case of mucoepidermoid carcinoma, and in one of five (20%) case of carcinoma ex pleomorphic adenoma. All cases were negative for polymerase chain reaction PCR-based analyses of BRAF mutations in codon 600. In studied salivary gland cancers, no PCR-based prove mutations of BRAF V600 were detected. Further molecular analyses are necessary to rapid molecular arrays for the identification of specific mutations, optimal for individualized targeted therapies.

Abstract LB-221: Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity, unique sequence homology and in vitro and in vivo antitumor efficacy

Abstract Siamab Therapeutics is developing monoclonal antibodies and antibody drug conjugates (ADCs) targeting tumor-associated carbohydrate antigens such as Sialyl-Tn (STn). These antibodies are selected using a proprietary glycan microarray that enriches for candidates whose binding is protein independent, highly specific and demonstrates exceptional target affinity. STn is a cancer specific antigen that is expressed on the surface of carcinomas including ovarian, colon, prostate, and pancreatic tumors but is rarely present in normal tissue. STn expression has been linked to innate immune suppression, a chemoresistant phenotype, metastasis, and poor prognosis. Previous attempts to target this antigen in the clinic with synthetic glycan vaccines proved safe but lacked efficacy. Siamab has developed over 20 murine mAbs that bind STn with high affinity and specificity. When formatted as ADCs, anti-STn mAb-ADCs target and selectively kill STn expressing cells and also demonstrate efficacy in multiple xenograft studies. Multiple murine mAbs perform well in IHC studies with multiple solid tumor samples and tissue microarrays and demonstrate high tumor specificity. Remarkable sequence homology across all murine mAbs was observed in both heavy and light chains, and hot spots for hypermutation were identified. Humanization was successfully completed using a selection of mAbs and multiple humanized mAbs maintain their target specific binding, affinity and cytotoxicity in vitro. Our data demonstrates that high-affinity, STn-specific mAbs show promise both as therapies for solid tumors and as a potential tool for the development of biomarkers and companion diagnostics enabling selection of patients most likely to respond to therapy. Citation Format: David Eaverone, Jeff Behrens, Jillian Prendergast. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity, unique sequence homology and in vitro and in vivo antitumor efficacy. [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 LB-221.

Abstract 4841: Molecular targets of NOSH-naproxen (AVT-219), a dual nitric oxide and hydrogen sulfide-releasing hybrid, in a xenograft mouse model of colon cancer

Abstract Introduction: Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colon cancer, but can cause serious gastrointestinal (GI), cardiovascular (CV), and renal side effects. Naproxen has a relatively favorable CV profile, but is notorious for its GI toxicity. To improve its safety profile, we developed NOSH-naproxen, where we linked nitric oxide (NO) and hydrogen sulfide (H2S) releasing moieties to the native molecule, the rational being the observations that NO and H2S may modulate some components of the local mucosal defense systems which should lead to reduced GI toxicity. Indeed, we recently reported that NOSH-naproxen displayed enhanced GI safety, with potent anti-inflammatory, analgesic, anti-pyretic, and anti-platelet properties. Here we describe the effects of NOSH-naproxen on the growth properties of several human colon cancer cell lines, and on its molecular targets in a xenograft mouse model of colon cancer. Methods: NOSH-naproxen was synthesized by us with 1H-NMR verification. Colon cancer cell lines: HT-29, HCT 15, and SW480; Cell growth: MTT. Xenografts: Male athymic nude (NU/NU) mice (N = 15) were implanted s.c. in the right flank with SW480 cells, when the tumors reached an average sizes of ∼100 mm3, the mice were randomly divided into 3 groups and gavaged daily with either vehicle (0.5% CMC) or equimolar concentrations of NOSH-naproxen (100 mg/kg) or naproxen (45 mg/kg). Tumor volume was measured every three days; after 30 days the mice were sacrificed, tumors collected, weighed, photographed and stored in formalin for IHC studies. Results: NOSH-naproxen inhibited the growth of HT-29, HCT 15, and SW480 cells with IC50 values of 90 ± 10, 100 ± 8, and 98 ± 7 nM at 24 hr, respectively. The corresponding IC50s for naproxen were 2800 ± 165, 2950 ± 215, and 3110 ± 185 nM. This represents an enhanced potency of ∼30,000-fold in the 3 cell lines at 24 h. In xenografts, all the mice treated with naproxen died within 2 weeks from what appeared to be GI bleeding. However, the NOSH-naproxen treated mice did not show any overt signs of toxicity. At sacrifice, NOSH-naproxen-treated mice had a mean reduction in tumor volume of 82%, and a mean reduction in tumor mass of 55%. NOSH-naproxen inhibited growth of these cancer cell xenografts as a result of reduced proliferation (decreased PCNA expression), and induction of apoptosis (increased number of TUNEL positive cells). NF-êB, activated in untreated xenografts was significantly inhibited by NOSH-naproxen. Other molecular targets affected by NOSH-naproxen were: induction of iNOS, significant reduction in FoxM1, and increases in p53. Conclusions: NOSH-naproxen is more potent and efficacious than naproxen and appears to be significantly safer. It targets parameters important in determining cellular kinetics, inflammation, and signaling pathways important in the carcinogenic process. Citation Format: Pascale L. Duvalsaint, Mitali Chattopadhyay, Ravinder Kodela, Khosrow Kashfi. Molecular targets of NOSH-naproxen (AVT-219), a dual nitric oxide and hydrogen sulfide-releasing hybrid, in a xenograft mouse model of colon cancer. [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 4841.

Pigmented epitheloid melanocytoma: a low grade/intermediate/ borderline melanoma

Pigmented epitheloid melanocytoma is a recently described entity, low grade malignant lesion, encompassing epitheloid blue nevus of carney complex and animal type melanoma, from which they are histologically indistinguishable. Excision biopsy of skin and lymph node were subjected to histopathological processing and IHC studies. The characteristic histomorphology and positive melanocytic marker in spindle, polygonal and epitheloid cellular components of both the skin and left supraclavicular lymph nodal lesions confirmed the diagnosis. We report a rare case of pigmented epitheloid melanocytoma of the anterior chest wall with metastasis to left supraclavicular lymph node.

AlgiMatrix™-Based 3D Cell Culture System as an In Vitro Tumor Model: An Important Tool in Cancer Research.

Routinely used two-dimensional cell culture-based models often fail while translating the observations into in vivo models. This setback is more common in cancer research, due to several reasons. The extracellular matrix and cell-to-cell interactions are not present in two-dimensional (2D) cell culture models. Diffusion of drug molecules into cancer cells is hindered by barriers of extracellular components in in vivo conditions, these barriers are absent in 2D cell culture models. To better mimic or simulate the in vivo conditions present in tumors, the current study used the alginate based three-dimensional cell culture (AlgiMatrix™) model, which resembles close to the in vivo tumor models. The current study explains the detailed protocols involved in AlgiMatrix™ based in vitro non-small-cell lung cancer (NSCLC) models. The suitability of this model was studied by evaluating, cytotoxicity, apoptosis, and penetration of nanoparticles into the in vitro tumor spheroids. This study also demonstrated the effect of EphA2 receptor targeted docetaxel-loaded nanoparticles on MDA-MB-468 TNBC cell lines. The methods section is subdivided into three subsections such as (1) preparation of AlgiMatrix™-based 3D in vitro tumor models and cytotoxicity assays, (2) free drug and nanoparticle uptake into spheroid studies, and (3) western blot, IHC, and RT-PCR studies.

Pathomorphological Diagnosis of Splenic Diffuse Red Pulp Small B-Cell Lymphoma

Background. Unclassifiable splenic B-cell lymphoma/leukemia is a rare and poorly studied disorder introduced in the WHO classification of hematopoietic and lymphoid tissue malignancies for the first time in 2008. This type of lymphoma requires differential diagnosing between hairy cell leukemia-variant (HCL-V) and splenic diffuse red pulp small B-cell lymphoma (SDRPL). Aim. To develop criteria for diagnosis of SDRPL by comparison of bone marrow biopsies (BMB) and surgical specimens of the spleen. Methods. In the Department of Morbid Anatomy of the Hematology Research Center, preoperative BMBs and surgical specimens of the spleen (2013-2015) were compared in 71 patients (men/women ratio 1:2.6, age range 44-81, median age 58 years) using morphological and extended immunohistochemical studies. Sanger sequencing and PCR assay were carried out to analyze the mutational status of IgHV and to identify mutations in MAP2K1, NOTCH, BRAF. Results. SDRPL was diagnosed in 5 (7 %) of 71 patients. In 2 groups of patients (with normal and high WBC count), the morphological features of spleen tissue were similar to those of a neoplastic substrate of HCL-V. The immunohistochemical assay demonstrates monomorphic expression of CD20 and DBA.44 and heterogeneous expression of CD11c, TRAP, CD103, CD123 in all cases. In none of the 5 cases, expression of CD25, CD27, Cyclin D1, Annexin-1 was found. In bone marrow (unlike HCL and HCL-V), predominantly interstitial and intravascular scant CD20+ lymphoid infiltration (4 of 5 cases) was found without detectable nucleoli in nuclei of small lymphoid cells. In 1 case, there was a combined lymphoid infiltration: CD20+ microfocalinterstitial infiltration with an intravascular component. No persistent molecular mutations in the spleen tissue specimens were found. Conclusion. SDRPL is diagnosed in 7 % of splenic B-cell lymphomas. It is a rare disorder, whose verification requires an integrated approach taking into account clini cal and laboratory data, results of flow cytometry, cytological, morphological, extended IHC and molecular biological studies.

Comparative study of core needle biopsy with excisional biopsy: To determine diagnostic accuracy & concordance with er/pr/her-2/neu status

Background: As there is continuous and increased need for early detection of cancer in breast lesion is never-ending. It is so, as the success of treatment depends upon early diagnosis. So role of core needle biopsy (CNB) in screening and diagnosing the breast lesion was evaluated. Aims and Objectives: To find concordance between the diagnostic power of, hormonal receptors, viz. estrogen receptors (ER), progesterone receptors (PR) sensitivity along with expression of HER-2/neu in CNB and excisional biopsy (EB) of breast lesion. Materials and Methods: We have studied the diagnostic accuracy of CNB in 60 breast lesion keeping the EB as gold standard. Immunohistochemical study (IHC) was simultaneously performed with three parameters as ER, PR and HER-2/neu. The results IHC studies of CNB were compared with the same study on EB specimens. Results: Out of which 39 cases were malignant and amazingly CNB has detected the malignancy in 38 cases. Also the IHC of ER, PR and HER-2/neu were well in concordance with the same study in EB specimen. Conclusion: Though CNB is excellent modality for screening, as study reflects, it can surely be used for confirmation also. As IHC results on CNB are also in well concordance with EB, the oncologist can lay out his treatment from very initial step of screening. Keywords: Core needle biopsy (CNB), Excisional biopsy (EB), Estrogen receptor (ER), Progesterone receptor (PR) and HER-2/neu

The Expression of P53 and ki67 in Colonic Polyps

Background: Colonic polyps are slow-growing overgrowth that projects above the surrounding mucosa's of the colon that carry a small risk (< 1%) of becoming malignant. However, because colonic polyps are highly prevalent in the general population (especially with increasing age), they confer an important predisposition to colon cancer and are therefore removed when detected. The distribution of Ki67 and p53 as markers of cell proliferation is very studied in Colorectal Carcinoma. In Iraq, IHC studies to detect p53 & ki-67 expression in GIT malignancy has been conducted. Aims of the study: To detect the expression of p53 & ki-67 in colonic polyps patient by immunohistochemistry. To assess the possible correlation between p53 &ki-67 in colonic polyps patient. To detect the possibility of conversion of pre-malignant colonic polyps to malignant form. Methodology: Fifty cases (27 male and 23 female ) were selected randomly with different types of colonic polyps were included in this study and using descriptive study. The age of patients were ranging from 4 up to 80 years, with mean age of [ 46.3 ] years. Cases were collected from the laboratory of histopathology in Al-sadder Medical city in Al-Najaf and from some private laboratories in the same governorate(Al-Najaf). the data analyzed by qi squared. Result: There is a significant correlation between P53 & age ,p53 & type of polyp also there is a significant correlation between Ki-67& age, Ki-67 & type of colonic polyp .There is a correlation between ki67 & p53. Conclusion: The study represent a trial to understand the probable conversion of polyps to colorectal carcinomaand the role of P53 & KI-67 in genesis of colorectal carcinoma. Recommendation: Screen and follow up to all patient with colonic polyps specially to patient with multiplepolyps. All type of polyps should be removed ,because polypectomy currently constitutes the best strategy forpreventing colorectal cancer

Intraoperative consultation of central nervous system lesions. Frozen section, cytology or both?

ObjectiveFrozen section is the traditional method of assessing central nervous system (CNS) lesions intraoperatively. Our aim is to determine the diagnostic accuracy of frozen section and/or cytological evaluation of CNS lesions in our center. Study designA total of 157 patients with CNS lesions underwent open surgical biopsy or excision in our center during a period of 2 years (2012–2013). All specimens were studied cytologically; of these specimens, 146 cases were also examined by frozen section. Cytology and frozen section slides were studied separately by two general pathologists who were blind to final diagnoses. The final diagnoses were based on permanent sections and IHC studies. ResultsThe accuracy rates of frozen section analysis and cytological evaluation were 87% and 86%, respectively. If the two methods were considered together, the accuracy rate improved to about 95%. ConclusionsCytological evaluation is an acceptable alternative to frozen section analysis and also a great supplement to the diagnosis of CNS lesions.

Utility Of Nitric Oxide And Hydrogen Sulfide-Releasing Chimeras As Anticancer Agents

Aspirin is chemopreventive but has significant side effects. We developed NOSH-aspirin a safer, nitric oxide and hydrogen sulfide releasing hybrid. Here we report on NOSH-aspirin as an anti-inflammatory and its effects on human cancer cell kinetics and various cancer xenografts. Anti-inflammatory: Carageenan rat paw edema model. Cancer cell lines: Colon, HT-29, HCT 15, SW 480; breast, MCF-7, MDA-MB-231; pancreas, MIA PaCa2, BxPC3. Normal cell lines: human mammary, HMEpC; pancreatic epithelial, ACBRI 515. Xenografts: nude mice implanted with HT-29, MDA-MB-231, MIA PaCa2 cells, were treated with NOSH-aspirin (100mg/kg/d) or vehicle. After 3 weeks, mice were sacrificed, tumors excised, weighed, and fixed for IHC studies. NOSH-aspirin significantly reduced paw edema as function of time. NOSH-aspirin's IC50 in nM at 24h for cell growth inhibition ranged from 50±2 to 250±10 in the cancer cell lines and about 400-fold higher in the normal cell lines. The cell growth inhibitory effects were due to a dose-dependent induction of apoptosis and cell cycle arrest (G0/G1), leading to reductions in cell proliferation. In xenografts, NOSH-aspirin had no effect on the weight of the mice. Tumor volume was reduced as a function of treatment time. At sacrifice, tumor mass reductions were colon: 89%, P=0.005; breast: 91%, P=0.006; pancreas: 75%, P=0.0031. Growth inhibition was due to reduced proliferation (decreased PCNA expression), and induction of apoptosis (increased TUNEL positive cells). NOSH-aspirin is a potent anti-inflammatory, preferentially affecting cancer cells and targets parameters important in determining cellular mass.

FBI-1 Is Overexpressed in Gestational Trophoblastic Disease and Promotes Tumor Growth and Cell Aggressiveness of Choriocarcinoma via PI3K/Akt Signaling

Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts.

Abstract P3-06-06: Prediction of benefit from endocrine therapy in ER+ early stage breast cancer: Correlative studies of the breast cancer index HoxB13/IL17BR (H/I) ratio, ER, PR, and HER2 expression in the randomized Stockholm trial

Abstract Background: Hormone receptor expression is an indication for endocrine therapy, however, receptor status is insufficient to account for the considerable heterogeneity of response, and additional predictive biomarkers are needed. The HoxB13/IL17BR (H/I) gene expression ratio has previously been shown to be predictive of benefit from extended endocrine therapy for women with estrogen receptor-positive (ER+) breast cancer treated in the MA.17 trial. In this correlative study, H/I was compared with ER, PR, and HER2 expression in assessing response to adjuvant tamoxifen (TAM) versus untreated (UNT) in ER+, node-negative patients from the prospective, randomized Stockholm trial. Methods: Analysis included 600 ER+, LN- patients (317 TAM, 283 untreated) from the Stockholm cohort. Expression profiles were generated by RT-PCR for H/I, ER, PR, and HER2, with additional IHC studies for ER, PR, and HER2. Multivariate Cox models including age, tumor size and tumor grade, were used to assess the significance of the interaction between treatment and each biomarker as continuous variables. 10-year risk of distant recurrence, as a function of each continuous biomarker, was estimated from Cox model in each of the 2 treatment arms. Results: The interaction between H/I and TAM treatment was significant (p = 0.003). Consistent with the significant interaction, the 10-year rate of distant recurrence as a function of continuous H/I demonstrated that the reduction in recurrence rates with TAM correlated with increasing H/I. Interaction P values for all other biomarkers were nonsignificant [ER (PCR), P=0.473; ER (IHC), p=0.371; PR (PCR), p=0.555; PR (IHC), p=0.475; HER2 (PCR), p=0.947, and HER2 (IHC), p=0.839]. The treatment effect of tamoxifen was unchanged across ER, PR, and HER2 expression levels. Conclusions: Results of this study provide further support for HoxB13/IL17BR as a biomarker of endocrine response. The H/I gene expression ratio, but not ER, PR, or HER2 expression evaluated as continuous variables, was predictive of benefit from adjuvant TAM treatment. Findings indicate that H/I endocrine response activity is not strictly correlated with ER+ expression and provides independent predictive information regarding estrogen signaling-driven recurrences. Citation Format: Yi Zhang, Brock E Schroeder, Piiha-Lotta Jerevall Jannok, Tommy Fornander, Olle Stal, Catherine A Schnabel, Dennis C Sgroi. Prediction of benefit from endocrine therapy in ER+ early stage breast cancer: Correlative studies of the breast cancer index HoxB13/IL17BR (H/I) ratio, ER, PR, and HER2 expression in the randomized Stockholm trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-06.

Paving the road to personalized medicine in cervical cancer: Theranostic biomarker evaluation in a 592-specimen library

Background: The results from Gynecologic Oncology Group protocol 240, that blockade and non-platinum chemotherapy, predictive biomarkers are requichemotherapy plus bevacizumab significantly improves overall survival over chemotherapy alone among women with advanced cervical cancer, represents a proof of concept of the potential for anti-angiogenesis therapy and the value of systemic therapy in this disease. To identify patients likely to derive the greatest benefit from angiogenesis red and predicated on theranostics, the emerging field through which developing technologies and capabilities in the diagnostic sector can be applied to pharmacogenomics and personalized medicine. Methods: We interrogated a database of theranostic biomarkers from the CARIS repository. 592 specimens in the cervical cancer library were evaluated by a combination of sequencing (NGS), gene amplification (ISH), and protein expression (IHC). Results: NGS sequencing in 224 specimens identified mutational hotspots corresponding to PI3KCA (26%), BRCA2 (21%), BRCA1 (10%), KRAS (10%), TP53 (10%), and FBXW7 (10%). Gene amplification of EGFR (11%, 20/174) and HER2 (8%, 32/395) was also observed. IHC studies were noteworthy for the following protein signatures: anti-programmed death receptor 1 (PD1) tumor infiltrating lymphocytes (65%, 53/82); over-expression of cMET (22%, 82/376); overexpression of estrogen receptor (20%, 118/590), progesterone receptor (8%, 48/589), and androgen receptor (4%, 22/578); gemcitabine-specific low RRM1 (44%, 256/538), topotecan-specific high TOPO1 (56%, 294/528), paclitaxelrelated high TLE3 (27%, 256/537) and low TUBB3 (74%, 202/273), and pemetrexed-related low TS (48%, 256/537). Conclusion: The August 14, 2014 U.S. FDA approval of bevacizumab for advanced cervical cancer constitutes a regulatory milestone that fulfills a high unmet clinical need. Our data support the inclusion of theranostic biomarkers to help guide therapy in clinical trials for patients who have progressed on antiangiogenesis therapy or who are considered otherwise incurable. Poly-ADPribose polymerase inhibition, MEK, cell cycle checkpoint, and PI3K/AKT/mTOR pathway inhibitors, EGFRand HER2-directed therapy, immunotherapy, hormonal therapy, and non-platinum chemotherapy may be suitable for study. Abstract (#3911)

Gastroenteropancreatic Neuroendocrine Tumours – An Institutional Experience

Objectives: 1)Tostudy thehistomorphological spectrum of gastroenteropancreatic neuroendocrine tumours(GEP NETs) and to analyze it`sclinicopathological features. 2) To classify the GEP-NETs as per WHO 2010 classification system.3) To analyze the expression of the NET marker Neuron specific enolase (NSE) in the above histomorphological spectrum.Methods: A retrospective study was done with collected cases of GEP-NET (over 7 year period) .Clinical data was collected from medical records.The histopathology slides were retrievedfrom archives and reviewed.Immunohistochemical (IHC) studieswere done with marker NSE.Results: The study included 11 cases of GEP-NETs. The age of the patients ranged from 13 to 65 years. The most common anatomic location was the appendix and colon with 3 cases each (27.27%). Most of the tumours were gradeI (6 cases). None of the tumours were associated with carcinoid syndrome. Six out of eleven cases had metastasis at time of diagnosis (54.54%). IHC studies done with NSEresulted in positivity seen in all 10 casesin which it was done.Conclusions: Gastroenteropancreatic neuroendocrine tumours are rare and occur over a wide age range. Most of these tumours present late with metastasis at time of diagnosis. The most common location of these tumours were in the appendix and proximal colon. NSE is a good marker for these tumours and is beneficial in confirming the diagnosis.