MIB-1 Staining Research Articles

AAA+ ATPases Reptin and Pontin as potential diagnostic and prognostic biomarkers in salivary gland cancer - a short report.

Salivary gland cancer (SGC) is a rare and heterogeneous disease with significant differences in recurrence and metastasis characteristics. As yet, little is known about the mechanisms underlying the initiation and/or progression of these diverse tumors. In recent years, the AAA+ ATPase family members Pontin (RuvBL1, Tip49a) and Reptin (RuvBL2, Tip49b) have been implicated in various processes, including transcription regulation, chromatin remodeling and DNA damage repair, that are frequently deregulated in cancer. The aim of this study was to assess the clinical and functional significance of Reptin and Pontin expression in SGC. Immunohistochemical staining of Pontin, Reptin, β-catenin, Cyclin D1, TP53 and MIB-1 was performed on a collection of 94 SGC tumor samples comprising 13 different histological subtypes using tissue microarrays. We found that Reptin and Pontin were expressed in the majority of SGC samples across all histological subtypes. Patients with a high Reptin expression showed a significantly inferior 5-year overall survival rate compared to patients with a low Reptin expression (47.7% versus 78.3%; p = 0.033), whereas no such difference was observed for Pontin. A high Reptin expression strongly correlated with a high expression of the proliferation marker MIB-1 (p = 0.003), the cell cycle regulator Cyclin D1 (p = 0.006), accumulation of TP53 as a surrogate p53 mutation marker (p = 0.042) and cytoplasmic β-catenin expression (p = 0.002). Increased Pontin expression was found to significantly correlate with both cytoplasmic and nuclear β-catenin expression (p = 0.037 and p = 0.018, respectively), which is indicative for its oncogenic function. Our results suggest a role of Reptin and Pontin in SGC tumor progression and/orpatient survival. Therefore, SGC patients exhibiting a high Reptin expression may benefit from more aggressive therapeutic regimens. Future studies should clarify whether such patients may be considered for more radical surgery, extended adjuvant therapy and/or targeted therapy.

Analysis of membranous Ki-67 staining in breast cancer and surrounding breast epithelium

Membranous Ki-67 staining with the MIB-1 antibody has been described in hyalinising trabecular adenomas of the thyroid and sclerosing haemangiomas of the lung. Its relatively rare occurrence in breast tumours has also been documented. The aim of the present study was to assess the rate of any membranous MIB-1 staining in breast specimens. The staining was performed at room temperature with 1:100 dilution of the antibody. One hundred four core needle biopsies and 41 operative specimens were analysed. Membranous staining was noted in 36/144 invasive carcinomas, 20/42 in situ carcinomas and 46/99 cases of peritumoural benign/normal breast epithelium. Most often, it presented as focal and partial polarised luminal membranous staining although complete circumferential staining also occurred, and membranous labelling was sometimes accompanied by cytoplasmic staining, too. In a few cases tested, greater dilution of the primary antibody did not abolish the membranous staining, which was absent with the SP6 monoclonal Ki-67 antibody. The membranous staining of invasive tumours showed no association with histological grade, lumen formation, oestrogen or progesterone receptor status or the Ki-67 nuclear labelling. In contrast, it was associated with a HER2-positive status, although it occurred in all molecular subtypes approached by immunohistochemistry. The background of this membranous staining remains elusive. It is unlikely to represent an artefact. At least partial sharing of an epitope of the nuclear Ki-67 protein with an unidentified membranous protein and some functional differences between membranous staining producing tumours and tumours lacking this pattern of staining may both contribute to some extent.

Evaluation of Ki-67 Index in Core Needle Biopsies and Matched Breast Cancer Surgical Specimens.

- The Ki-67 index is strongly prognostic and is used as a surrogate marker to distinguish luminal A from luminal B breast cancer types. - To investigate differences in Ki-67 index between core needle biopsy samples and matched surgical samples in breast cancer. - We included patients with invasive breast cancer who did not receive neoadjuvant therapy. A total of 89 pairs of core needle biopsies and surgical specimens were collected, and the Ki-67 index was assessed in hot spot areas using an image analyzer. We applied a 14% Ki-67 index to define low versus high groups. - The Ki-67 index was significantly higher in core needle biopsies than in surgical specimens ( P < .001), with a median absolute difference of 3.5%. When we applied 14% as a cutoff, 16 of 89 cases (18%) showed discrepancy. Thirteen cases showed a high Ki-67 index in core needle biopsies but a low Ki-67 index in surgical samples. There were 10 cases (11.2%) that showed discordant luminal A/B types between core needle biopsy and the matched surgical specimen. The reasons for the discordance were poor staining of MIB1 accompanied by fixation issues and intratumoral heterogeneity of the Ki-67 index. - A significant difference in the Ki-67 index between core biopsy and surgical specimens was observed. Our findings indicate that it may be better to perform the Ki-67 assay on the core needle biopsy and the surgical specimen than on only one sample.

Induced Granulation Tissue but not Artificial Dermis Enhances Early Host-Graft Interactions in Full-Thickness Burn Wounds.

Cellular grafts used for skin repair require rapid integration with the host tissue to remain viable and especially to nourish the epidermal cells. Here, we evaluated the responses in the split-thickness skin grafts (STSGs) grafted on three differently treated wound beds: directly on excised wound bed (EX), on an artificial dermal template (DT) and on granulation tissue (GT) induced by cellulose sponge. In ten burn patients, after excision, a test area was divided into three sections: One transplanted with STSG instantaneously and two sections had a pre-treatment for 2weeks with either DT or a cellulose sponge inducing granulation tissue formation and thereafter grafted with STSGs. One week after grafting, the STSGs on GT demonstrated most endothelial CD31+ staining, largest average vessel diameters as well as most CD163+ staining of M2-like macrophages and most MIB1+ proliferating epidermal cells, suggesting an active regenerative environment. STSGs on DT had smallest vessel diameters and the least CD163+ macrophages. STSGs on EX had the least CD31+ cells and the least MIB1+ proliferating cells. After 3months, this reactivity in STSGs had subsided, except increased dermal cell proliferation was observed in STSGs on EX. Results show that pre-treatment of wound bed and induction of granulation tissue formation can accelerate host-graft interaction by stimulating graft vasculature and inducing cell proliferation.

Anti-Tumour Effects of High Affinity L-type Amino Acid Transporter1 Inhibitors on Human Pancreatic Adencarcinoma Cells

Background: a system L transporter L-type amino acid transporter 1 (LAT1) is upregulated to support tumor cell growth in malignant tumor. Objective: The purpose of the present study was to investigate the growth inhibition and [14C] L-leucine transport in human pancreatic adenocarcinoma cells MAIPaCa-2 and BXPC3. Methodology: This animal study was carried out in Japan. The in vitro growth inhibition study was performed by using KYT0351 and KYT0353 which inhibited the tumor cell growth in dose dependent manner and uptake of [14C] L-leucine by MIAPaCa-2 and BXPC3 cells was Na+- independent and was strongly inhibited by KYT0351 and KYT0353. The in vivo tumor growth inhibition was also carried out by intra tumor injection of KYT0351 and KYT0353 at the concentration of 2.6mM of each on both the MIAPaCa-2 and BXPC3 nude mice tumor. Result: In a subsequent survival study with the intra peritoneal injection of ascites mice model, control mice had a mean life span of 20 ± 4.30 days and 21 ± 5 days in MIAPaCa-2 and BXPC3 cells respectively, whereas the intraperitoneal injection of 10mg/kg twice daily of KYT0351 and KYT0353 group had improved survival (mean life span 28.4 ± 8.5 and 34.4 ± 9.86 days, 26 ±4.52 and 31.8 ± 7.62 days respectively in MIAPaCa-2 and BXPC3 cells). Kaplan-Meier survival data of nude mice treated with KYT0351 and KYT0353 were significant. To study the mechanism of growth inhibition we investigated the MIB-1 proliferation assay and TUNEL assay. Significantly less MIB-1 staining and more apoptotic nuclei was detected in tumors treated with KYT0351 and KYT0353 in both MIAPaCa-2 and BXPC3 cells compared with saline treated group. Conclusion: In conclusion both the KYT0351 and KYT0353 is a potent LAT1-specific inhibitor and LAT1 could be one of the molecular target in pancreatic adenocarcinoma therapy. Journal of National Institute of Neurosciences Bangladesh, 2016;2(2): 55-68

Prognostic value of WHO grade in pancreatic neuro-endocrine tumors in Multiple Endocrine Neoplasia type 1: Results from the DutchMEN1 Study Group

BackgroundThe prognostic value of WHO grade in pancreatic neuroendocrine tumors (PanNETs) in patients with Multiple Endocrine Neoplasia Type 1 (MEN1) is unknown. MethodsWe performed a cohort study using the Dutch National MEN1 database, which includes >90% of the Dutch MEN1 population with data collected between 1990 and 2014. Formalin-fixed paraffin embedded tissue blocks from the largest resected PanNET per patient were collected. MIB1 staining was performed and KI67 labeling index (LI) was determined by manual eye-counting under a microscope and by digital image analysis. Mitotic count was evaluated from hematoxylin & eosin stains. Association between WHO grade and (time until) development of liver metastases was calculated. ResultsSixty-nine MEN1 patients who underwent pancreatic surgery were included. Ten patients (14%) developed liver metastases and all had PanNETs ≥3 cm. WHO G1, G2 and G3 PanNETs were seen in 83% (n = 57), 16% (n = 11) and 1% (n = 1) respectively. In non-functioning PanNETs >2 cm, liver metastases occurred in 80% of WHO G2 PanNETs (4/5) compared to 23% (5/22) in WHO G1 PanNETs (p = 0.03) when WHO grade was based on mitotic count only. This significant association was not seen for WHO grade based on Ki67 LI. After five years, liver metastases in non-functioning PanNETs were not seen in tumors ≤2 cm, in 10% of the large WHO G1 (according to mitotic count only) tumors and in 60% of large WHO G2 tumors (p-value 0.000). ConclusionHigh mitotic count is correlated with poor prognosis in MEN1 patients with large non-functioning PanNETs.

BRCA1 expression, proliferative and apoptotic activities in ovarian epithelial inclusions

BackgroundThe purpose of this study was to examine proliferative and apoptotic activity in relation with BRCA1 expression in ovarian epithelial inclusions (OEIs), the putative precursor lesions of ovarian epithelial cancer (OEC).MethodsBenign ovaries from 95 patients were examined. Dual immunohistochemical staining for both BRCA1 and MIB-1 were performed to examine the relationship between BRCA1 and MIB-1 in OEI cells. Apoptotic activity was assessed on the parallel tissue sections by using TUNEL assay. Patients’ age, menstrual phase and menopausal status were analyzed.ResultsOEIs were present in the ovaries of 53% of the patients. OEIs were less frequently found in premenopausal (45%) than postmenopausal women (58%). BRCA1 and MIB-1 were found in 27 and 47% of the OEI-containing ovaries, respectively. All BRCA1 positive OEI cells are MIB-1 positive with dual staining method, although overall the percentage of positive cells was small. No significant difference was found for BRCA1 and MIB-1 expression in OEIs between menopausal status and menstrual phases. Apoptosis containing OEIs were seen in 70% of the ovaries. Compared to OEIs in proliferative menstrual phase and premenopausal status, significantly more apoptosis was found in OEIs from secretory phase and postmenopausal women. A small fraction of the epithelial cells within OEIs are proliferating or dying.ConclusionsLow estrogen and/or high progesterone levels may promote OEI cell turnover via induction of apoptosis. Imbalance between cell proliferation and death within OEIs under influence of hormones may play a role in the ovarian epithelial tumorigenesis.

In stage pT1 non-muscle-invasive bladder cancer (NMIBC), high KRT20 and low KRT5 mRNA expression identify the luminal subtype and predict recurrence and survival.

Differential expression of cytokeratins (CK) is a characteristic feature of chemoresistant luminal (KRT20) and chemosensitive intrinsic aggressive basal (KRT5) subtypes in muscle-invasive bladder cancer (MIBC). We investigated mRNA expression of KRT5 and KRT20 and its predictive value in stage pT1 bladder cancer. In retrospective analysis of clinical data and formalin-fixed paraffin-embedded tissues (FFPE) of patients with stage pT1 NMIBC who underwent transurethral resection of the bladder, a single-step RT-qPCR was used to measure mRNA expression. Furthermore, immunohistochemical (IHC) staining of CK20, panCK, and MIB1 was performed. Valid measurements were obtained from 231 samples out of a series of 284 patients. Spearman correlation revealed significant associations between mRNA and protein expression of KRT20/CK20 (ρ 0.6096, p<0.0001) and MKI67/MIB1 (ρ 0.5467, p<0.0001). A positive correlation was found between MKI67 and KRT20 expression (ρ 0.3492, p<0.0001), while MKI67 and KRT5 were negatively correlated (ρ -0.1693, p=0.01). High KRT20 expression (≥40.26) was significantly associated with worse recurrence free survival (RFS) (p=0.001), progression-free survival (PFS) (p=0.0003), and cancer specific survival (CSS) (p=0.0414). The combination of high KRT20 expression and low KRT5 expression (<36.83) was associated with unfavorable RFS (p=0.0038) and PFS (p=0.0003) and proved to be the only independent predictor for RFS (p=0.0055) and PFS (p=0.0023) in multivariate analysis. KRT20 mRNA determination was superior to CK20 protein estimation with regard to RFS and PFS prediction. KRT20 and KRT5 mRNA quantification can predict recurrence and progression of stage pT1 NMIBC reflecting basal and luminal subtypes of MIBC and is superior to CK20 protein expression determined by IHC.

Rare Case of Painless Jaundice

Cervical cancer remains a major cause of morbidity and mortality. The vast majority are related to human papillomavirus infection which is ubiquitous in the population and results in approximately 12,710 new cases and 4,290 deaths in the US annually, making it the third most common gynecological cancer diagnosis and cause of death. Squamous cell carcinoma and adenocarcinoma account for approximately 69% and 25% of cases of cervical carcinoma, respectively. Cervical cancer may spread to adjacent organs via direct extension or to distant sites through lymphatic or hematogenous dissemination. The most common sites of hematogenous spread are the lung, liver, and bone. Here we present a case of cervical cancer with metastasis to the biliary system without liver metastasis causing common bile duct obstruction with jaundice. A 63-year-old woman with history of cervical cancer presented with painless jaundice. She was diagnosed four months prior with stage IV-A cervical carcinoma and underwent chemotherapy with cisplatin and whole pelvic radiation. After completing five courses of brachytherapy, the last of which was one month prior to admission, she was determined to be in clinical remission. Upon admission, MRCP was obtained and demonstrated evidence of biliary obstruction. ERCP then showed high-grade stenosis of the proximal CBD and beading of the intrahepatic bile ducts, which was treated with stenting. ERCP brushings confirmed metastatic disease with atypical epithelial cells consistent with metastatic SCC. Immunohistochemical studies demonstrated strong and diffuse cytoplasmic and nuclear p16 staining while MIB-1 staining was present in 40-50% of the neoplastic cells. Metastatic cervical carcinoma is a devastating disease with a grim prognosis. Although the cancer typically spreads by direct extension, distant metastases to the lung, liver, and bone have been described. Per our literature review, there has been a single case of metastasis to the ampulla of Vater but none to the bile ducts itself. This patient with a history of stage IV-A cervical carcinoma presented with painless jaundice and was subsequently diagnosed with bile duct metastasis. Unfortunately, the patient was not a candidate for surgical or chemotherapy treatment and was sent to hospice due to her functional status. Fortunately, the incidence and mortality rate of cervical cancer has declined in recent years in the US due to increased cervical cancer screening vigilance.Figure 1Figure 2Figure 3

The Relationship between Expression of p70s6k with Radiotherapy Response in Cervical Cancer

There are many important prognostic factors in advanced stage cervical cancer primary treated with radiotherapy. Besides clinical factors, many biomarkers have been studied in relation with radiotherapy response. The p70s6k is a biomarker which plays a significant role in cell proliferation. Increased levels of p70s6k are also associated with drug resistance in cancer. In the present study, the relationship between the expression level of p70s6k before treatment and the radiotherapy response in cervical cancer was determined. Immunohistochemical staining of p70s6k, MIB-1, and p53 was conducted in microscopic slide from 21 cancer tissue biopsies before treatment with radiotherapy. After the treatment had completed early radiotherapy response was observed by pelvic control method. The results showed that p70s6k is partly highly expressed (61.9%, 13/21) and partly low expressed (38.1%, 8/21) in the cancer cells. They also show that there is no statistically significant differences on AgNOR mean, MIB-1, and p53 indices in the different degrees of p70s6k expression (p≥0.05). Higher expression levels of p70s6k is more strongly associated with good radiotherapy response compared to lower ones (p=0.05). In conclusion, the level of p70s6k expression before treatment has an association with radiotherapy response.

Defining a New Prognostic Index for Stage I Nonseminomatous Germ Cell Tumors Using CXCL12 Expression and Proportion of Embryonal Carcinoma.

Up to 50% of patients diagnosed with stage I nonseminomatous germ cell tumors (NSGCTs) harbor occult metastases. Patients are managed by surveillance with chemotherapy at relapse or adjuvant treatment up front. Late toxicities from chemotherapy are increasingly recognized. Based on a potential biologic role in germ cells/tumors and pilot data, our aim was to evaluate tumor expression of the chemokine CXCL12 alongside previously proposed markers as clinically useful biomarkers of relapse. Immunohistochemistry for tumor expression of CXCL12 was assessed as a biomarker of relapse alongside vascular invasion, histology (percentage embryonal carcinoma), and MIB1 staining for proliferation in formalin-fixed paraffin-embedded orchidectomy samples from patients enrolled in the Medical Research Council's TE08/22 prospective trials of surveillance in stage I NSGCTs. TE08/TE22 trial patients had a 76.4% 2-year relapse-free rate, and both CXCL12 expression and percentage embryonal carcinoma provided prognostic value independently of vascular invasion (stratified log rank test P = 0.006 for both). There was no additional prognostic value for MIB1 staining. A model using CXCL12, percentage embryonal carcinoma, and VI defines three prognostic groups that were independently validated. CXCL12 and percentage embryonal carcinoma both stratify patients' relapse risk over and above vascular invasion alone. This is anticipated to improve the stratification of patients and identify high-risk cases to be considered for adjuvant therapy.

Fluorescence in situ hybridization for chromosome 14q deletion in subsets of meningioma segregated by MIB-1 labelling index.

To correlate histopathological grading of meningiomas segregated into subgroups based on the MIB-1 labelling index (MIB-1 LI) with chromosomal loss of 14q using fluorescence in situ hybridization (FISH). Retrospective study conducted in a tertiary hospital. Forty-six cases from January to December 2011 were segregated into 5 categories based on the MIB-1 LI. Slides were reviewed to ascertain the grade. Immunohistochemical staining for MIB-1 was performed using a Ventana Benchmark XT autostainer. Commercially available FISH paraffin pretreatment kit and SpectrumOrange fluorophore labelled probe were used. The Statistical Package for the Social Sciences version 16.0 for Windows was used for statistical analysis. There were 21 World Health Organisation (WHO) grade I, 24 grade II, and 1 grade III meningiomas. There was a statistically significant difference between the mean duration of symptoms, maximum dimension, and the MIB-1 LI of grade I and grade II meningiomas. 33.3% grade I cases showed 14q deletion, compared to 84% of grade II and III meningiomas. Histologically, hypercellularity, small cell formation, prominent nucleoli, and sheet-like growth were significantly associated with 14q deletion. All brain invasive meningiomas had 14q deletion. As MIB-1% increased, the prevalence of deletions was significantly higher. The mean MIB-1 of the 7 grade I meningiomas that had 14q deletions was 8.86 ± 1.95% when compared to 4.14 ± 1.35% for those without 14q deletions. A strong association existed between histologic grade, MIB-1 LI, and the presence of chromosome 14q deletion. Association of high MIB-1 LI with 14q deletions, even in meningiomas with a Grade I histology, defines a distinct subset of benign meningiomas.

Abstract C145: Targeted radiosensitization of non-small cell lung cancer (NSCLC) through ADAM17 inhibition

Abstract Introduction: Radiotherapy is a mainstay for the treatment of locally advanced NSCLC. However, the response rates and clinical outcomes are still disappointing, with the 5-year survival rate being only approximately 15%. Growth and survival of NSCLC cells are often dependent on ectodomain cleavage of membrane-anchored proteins, which is primarily mediated by metalloproteases such as ADAMs (a disintegrin and metalloproteinase). Among all metalloproteases, ADAM17 is actively associated with this process of proteolytic ‘shedding’ and hence the release of growth factors and cytokines regulating cell proliferation and migration. In the current study, we provide novel insights into activation of ADAM17 and subsequent ligand shedding in response to irradiation. We show for the first time that direct targeting of ADAM17 with a clinically relevant inhibitor radiosensitizes lung carcinoma in vitro and in vivo. Material and methods: Large scale secretome profiling (&amp;gt;300 factors) was performed using antibody arrays for a wide range of secretory factors. Secretion kinetics of selected ADAM17 substrates were determined using ELISA across different established tumor cells and in murine blood serum, derived from irradiated A549 tumor xenograft-carrying mice. Clonogenic survival and xenograft tumor growth delay assays were performed in response to IR in siRNA-targeted tumor cell lines or in combination with small molecular agents. Tumor proliferation and ADAM17 downstream was evaluated employing immunohistochemistry and ex vivo blood serum analysis. Results: Based on a large scale secretome screening, we investigated secretion of auto- or paracrine factors in non–small cell lung cancer in response to irradiation and discovered the ADAM17 network as crucial mediator of resistance to IR. Irradiation triggered secretion of multiple ADAM17 substrates as ALCAM and amphiregulin in a similar IR-induced time- and dose-dependent manner across a panel of NSCLC cell lines and from A549-derived tumor xenografts. Irradiation also induced a dose-dependent increase of furin-mediated cleavage of the proform of ADAM17 to active ADAM17, which resulted in enhanced ADAM17 activity in vitro and in vivo. Genetic or pharmacologic targeting of ADAM17 suppressed IR-induced shedding of secreted factors, downregulated ErbB-signaling in target cells and enhanced IR-induced cytotoxicity in vitro. Furthermore, the combined treatment modality of IR with the ADAM17 inhibitor TMI-005 resulted in a supra-additive antitumor response in A549 tumor xenografts. Inhibition of ADAM17 with TMI-005 also strongly reduced immunohistochemical staining for ADAM17 substrates and MIB1, demonstrating an enduring antiproliferative effect and potency of ADAM17 targeting in combination with radiotherapy to overcome treatment resistance. Conclusions: These findings implicate that radiotherapy significantly activates ADAM17 in non–small cell lung cancer (NSCLC) cells, which results in shedding of multiple survival factors, growth factor pathway activation and contributes to treatment resistance. We provide a sound rationale for repositioning ADAM17 inhibitors as short-term adjuvants to improve the radiotherapy outcome of NSCLC. Citation Format: Ashish Sharma, Sabine Bender, Oliver Riesterer, Angela Broggini-Tenzer, Martin Pruschy. Targeted radiosensitization of non-small cell lung cancer (NSCLC) through ADAM17 inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C145.

Interlaboratory variability of MIB1 staining in well-differentiated pancreatic neuroendocrine tumors.

Neuroendocrine tumors (NET) are routinely graded and staged to judge prognosis. Proliferation index using MIB1 staining has been introduced to assess grading. There are vivid discussions on cutoff definitions, automated counting, and interobserver variability. However, no data exist regarding interlaboratory reproducibility for low proliferation indices which are of importance to discriminate between G1 and G2 NET. We performed MIB1 staining in three different university hospital-based pathology laboratories on a tissue micro array (TMA) of a well-characterized patient cohort, containing pancreatic NET of 61 patients. To calculate the proliferation index, number of positive tumor nuclei was divided by the total number of tumor nuclei. Labeling index was compared to mitotic counts in whole tissue sections and to clinical outcome. Linear regression analysis, intraclass comparison, and log-rank analysis were performed. Intraclass correlation showed moderate-to-fair agreement. Especially low proliferating tumors were affected by interlaboratory differences. Log-rank analysis was performed for each lab and resulted in three different cutoffs (5.0, 3.0, and 0.5 %). Every calculated cutoff stratified the patient cohort to a significant extent for the underlying stain (p < 0.001, <0.001, and <0.001) but showed no or lesser significance when applied to the other stains. Significant and relevant interlab differences for MIB1 exist. Since the MIB1 proliferation index influences grading, local cutoffs or external standardization should urgently be introduced to achieve reliability and reproducibility.

AI-21 * HIF-1A TARGETING IN AN ORTHOTOPIC XENOGRAFT MODEL THAT RECAPITULATES THE HYPOXIC MICROENVIRONMENT OF GLIOBLASTOMA

INTRODUCTION: Tissue hypoxia and necrosis represent a pathological hallmark of glioblastoma. Hypoxia inducible factor 1a (HIF-1a) plays crucial roles in the malignant phenotypes of glioblastoma, and may offer a therapeutic target. However, developing HIF-1a targeted agents has been hampered by the lack of a preclinical model that recapitulates the disease relevant hypoxic microenvironment. MATERIALS AND METHODS: MGG123 neurosphere culture was established from a surgical specimen of a recurrent glioblastoma. Tumorigenicity of MGG123 cells was assessed by orthotopic xenografting in SCID-mice. The original tumor and xenografts were characterized by H&E staining and immunohistochemistry for HIF-1a, MIB-1, CD34, nestin and CD44. Hypoxic foci within xenografts were detected by pimonidazole immunofluorescence. In vitro, HIF-1a expression was evaluated by Western blot under normoxia and hypoxia after treatment with Digoxin and Ouabain, shown to inhibit HIF-1a synthesis. In vivo, mice bearing intracerebral MGG123 tumors were treated with intraperitoneal injections of digoxin, and its effects on HIF-1a expression, CD34+ vasculature, and overall survival were examined. RESULTS: MGG123 cells generated aggressive orthotopic xenografts that contain foci of pseudopalisading necrosis. The patient and xenografted tumors showed histopathological similarities as they both have high MIB-1 indices and are diffusely positive for CD44 and nestin. HIF-1a immunopositivity was specifically found in peudopalisading cells surrounding necrosis in both tumors. Within the xenografts, hypoxic areas were never found within 50mm proximity to CD34+ blood vessels. Hypoxia enhanced HIF-1a expression in cultured MGG123, and this was abrogated by treatment with Digoxin or Ouabain. In vivo, digoxin decreased HIF-1a expression and CD34+ vasculature within orthotopic MGG123 tumors, and extended overall survival of brain tumor-bearing mice. CONCLUSION: We established a preclinical glioblastoma model that uniquely mirrors the hypoxic microenvironment of human glioblastoma, and is suitable for developing HIF-1a targeted agents. Digoxin down-regulated HIF-1a and angiogenesis and increased overall survival in this clinically relevant glioblastoma model.

Hyaline matrix in hyalinizing trabecular tumor

Hyalinizing trabecular tumor (HTT) is a rare neoplasm which usually follows an indolent clinical course. The cytologic diagnosis of HTT can be challenging as these neoplasms share cytomorphological features with other thyroid neoplasms and paraganglioma. In fine-needle aspiration (FNA) smears a diagnosis of papillary thyroid carcinoma (PTC) or suspicion of PTC is often made. Herein we report cytologic findings in two patients with HTT examined by FNA. The key to a correct diagnosis is the recognition of a hyaline and colloid/amyloid-like material in the background of the smears. Immunocytochemical examination showing aberrant membranous and peripheral cytoplasmic staining for MIB-1 can help in rendering a correct diagnosis.

Abstract 3484: Ex-vivo cultures of freshly explanted tumor specimens (TIPCAN®), a potent translational approach for screening novel targeted agents

Abstract Background: In vitro cell lines and in vivo mouse models are important screening tools for novel anticancer agents. However those preclinical models do not reliably reflect the complexity of human tumors and are, by essence, incapable of evaluating the intricate interplay of cancer cells in their native stroma. To evaluate novel drugs in hepatocellular (HCC) and Head and Neck squamous cell (HNSCC) carcinomas, we developed TIPCAN® as a novel approach using freshly explanted human tumor slices to test the pharmacodynamic effects of targeted agents or classic chemotherapies. Materials and Methods: Tumor samples were obtained from fresh surgical specimens and biopsies and cut in 300 µm thick slices using a Leica microtome. TIPCAN® was applied on tumor slices that were maintained in culture for 24-72 hours in a defined environment that allows for diffusion of oxygen and nutrient, in a specific culture medium. Viability of tissues was evaluated using different methods. Individual HCC and HNSCC tumor slices were exposed to various concentrations of targeted agents or chemotherapies. Tumor samples were then analyzed by immunohistochemistry and immunofluorescence for various pharmacodynamics. Slides were digitalized using an APERIO Scanscope system and biomarker expression was quantified using Image J Software. Results: We first established the suitable conditions for ex vivo culture, by testing several O2 levels and medium compositions. We then quality controlled TIPCAN® using over 30 liver tumors and defined HE staining as the most reliable tool to assess tissue quality and viability. Using appropriate biomarkers, we also confirmed that 24 to 72 hours of culture had minimal consequences on the potential phenotypical drift of HCC tumor slices, confirming the steadiness of our model, and allowing us to perform pharmacodynamics evaluation of several therapies. As an example, using our ex vivo approach we evaluated the consequence of sorafenib exposure on HCC tumors explants. We showed that sorafenib decreased HCC proliferation by almost 70%, induced a 2 to 8-fold increase in apoptosis and inhibited by 80% ERK and AKT phosphorylation, mimicking the reported effects of sorafenib in vivo. We then translated our technology in HNSCC, in which we tested the combination of carboplatin and everolimus and compared our results with clinical trial patients treated with the same drugs (CAPRA trial). Carboplatin and everolimus combination showed a decrease of S6 phosphorylation and MIB1 staining, and induction of caspase3, mimicking the effects observed in patients. Conclusion: Taken together, our results show that TIPCAN® can be used as a valuable tool to assess the pharmacodynamic effects of anticancer agents in HCC and HNSCC. TIPCAN® could contribute to evaluate drug effects in &amp;lt;72h in fresh human tumor tissues and could therefore be helpful for clinicians to select patients with HCC and HNSCC for intelligence medicine. Citation Format: Annemilai Tijeras-Raballand, Maria Serova, Miguel Albuquerque, Nathalie Colnot, pierre Bourgoin, Nelly Müller, Safi Dokmak, Mohamed Bouattour, Jacques Belghiti, Eric Raymond, Sandrine Faivre, Valérie Paradis, Armand de Gramont. Ex-vivo cultures of freshly explanted tumor specimens (TIPCAN®), a potent translational approach for screening novel targeted agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3484. doi:10.1158/1538-7445.AM2014-3484

Colitis cystica profunda indefinite for dysplasia in Crohn disease: A potential diagnostic pitfall

Colitis cystica profunda (CCP) is a nonneoplastic condition characterized by misplaced glands deep to the muscularis mucosae of the colon and may be difficult to differentiate from well-differentiated mucinous adenocarcinoma. Absence of dysplasia in CCP usually aids in this distinction. We present a challenging case of CCP in the setting of Crohn disease (CD) containing foci of atypical epithelium. A right hemicolectomy from a 46-year-old woman contained a stricture associated with a proximal multilocular cystic lesion containing mucin-filled glands dissecting through the colonic wall. These glands had lobulated architecture with smooth contours surrounded by lamina propria and lacking desmoplastic stroma. The epithelium had focal nuclear crowding, enlargement, and hyperchromasia, with increased nucleus to cytoplasm ratio, but overall preserved polarity. Atypical cells were focally positive for CK7 and p53, with increased MIB-1 staining. These findings were interpreted as indefinite for dysplasia. Chronic transmural inflammation and mucosal regeneration probably facilitated epithelial misplacement, which secondarily developed cytologic atypia. However, the overall architecture and lack of dysplasia in the overlying mucosa argue against a diagnosis of adenocarcinoma. Our case illustrates the difficult diagnosis of this uncommon but problematic phenomenon, awareness of which is paramount for pathologists and clinicians participating in the management of CD patients.

Correlation of Clinicopathologic Parameters and Immunohistochemical Features of Triple-negative Invasive Lobular Carcinoma

Invasive lobular carcinoma (ILC) is a subtype of invasive breast carcinoma. With the advent of gene profiling, breast cancer has been classified into luminal A, luminal B, HER2-overexpressing, and triple-negative carcinoma (TNC). Several studies have described TNC (ER, PR, HER2) as a surrogate for basal-like breast carcinoma. However, there is sparse literature on triple-negative lobular carcinoma (TNLC), as most of them show hormone receptor expression. The aim of this study was to investigate the correlation of clinicopathologic parameters of TNLC that has been demonstrated in invasive ductal carcinoma. Clinicopathologic parameters and immunohistochemical stains for ER, PR, E-cadherin, HER2, MIB1, and fluorescent in situ hybridization for HER2 of 255 ILC cases were retrieved. In addition, immunohistochemical analysis was performed for p53, c-kit, vimentin, p16, cyclinD1, and BCL2 on 78 cases where 12 were TNC cases and 66 were non-TNC cases. Of the 255 ILC cases, 218 (85.5%) were classic and 37 (14.5%) were pleomorphic. Seventy-seven (30.1%) cases showed axillary lymph node metastasis. There were 14 of the 255 TNC cases (5.49%) that showed higher incidence in the elderly patients. Six of the 37 (16.21%) cases were pleomorphic and 8 of the 218 (3.7%) cases were classic. Positivity for vimentin was seen in 8 of the 12 cases (67.7%), CK 5 in 3 of the 12 (25%) cases, p16 in 11 of the 12 (91.6%) cases, p53 in 8 of the 12 (66.7%) cases, c-kit in 6 of the 12 (50%) cases, and cyclinD1 in 6 of the 12 cases (50%) indicating basal-like phenotype in 3 cases and nonbasal-like phenotype in 9 cases. There was no statistical significance in lymph node metastasis, tumor recurrence, and distant metastasis between TNC and non-TNC. TNLC showed distinct clinicopathologic features such as more frequently seen in the elderly, pleomorphic, larger tumor size, increased expression of vimentin, CK 5, p16, p53, and c-kit. Not all cases showed basal-like phenotype. TNLC is less frequently seen as compared with TNC in invasive ductal carcinoma.

Association of human papilloma virus with atypical and malignant oral papillary lesions

This study aimed to examine atypical and malignant papillary oral lesions for low- and high-risk human papillomavirus (HPV) infection and to correlate HPV infection with clinical and pathologic features. Sections of 28 atypical papillary lesions (APLs) and 14 malignant papillary lesions (MPLs) were examined for HPV by in situ hybridization and for p16 and MIB-1 by immunohistochemistry; 24 conventional papillomas were studied for comparison. Low-risk HPV was found in 10 of 66 cases, including 9 APLs and 1 papilloma. All low-risk HPV-positive cases showed suprabasilar MIB-1 staining, and the agreement was statistically significant (P < .0001). Diffuse p16 staining combined with high-risk HPV was not seen in any of the cases. A subset of HPV(-) APLs progressed to carcinoma. Oral papillary lesions are a heterogeneous group. Low-risk HPV infection is associated with a subset of APLs with a benign clinical course. Potentially malignant APLs and MPLs are not associated with low- or high-risk HPV.