Formalin-fixed Paraffin-embedded Tissue Microarrays Research Articles

An N-glycome tissue atlas of 15 human normal and cancer tissue types determined by MALDI-imaging mass spectrometry

N-glycosylation is an abundant post-translational modification of most cell-surface proteins. N-glycans play a crucial role in cellular functions like protein folding, protein localization, cell–cell signaling, and immune detection. As different tissue types display different N-glycan profiles, changes in N-glycan compositions occur in tissue-specific ways with development of disease, like cancer. However, no comparative atlas resource exists for documenting N-glycome alterations across various human tissue types, particularly comparing normal and cancerous tissues. In order to study a broad range of human tissue N-glycomes, N-glycan targeted MALDI imaging mass spectrometry was applied to custom formalin-fixed paraffin-embedded tissue microarrays. These encompassed fifteen human tissue types including bladder, breast, cervix, colon, esophagus, gastric, kidney, liver, lung, pancreas, prostate, sarcoma, skin, thyroid, and uterus. Each array contained both normal and tumor cores from the same pathology block, selected by a pathologist, allowing more in-depth comparisons of the N-glycome differences between tumor and normal and across tissue types. Using established MALDI-IMS workflows and existing N-glycan databases, the N-glycans present in each tissue core were spatially profiled and peak intensity data compiled for comparative analyses. Further structural information was determined for core fucosylation using endoglycosidase F3, and differentiation of sialic acid linkages through stabilization chemistry. Glycan structural differences across the tissue types were compared for oligomannose levels, branching complexity, presence of bisecting N-acetylglucosamine, fucosylation, and sialylation. Collectively, our research identified the N-glycans that were significantly increased and/or decreased in relative abundance in cancer for each tissue type. This study offers valuable information on a wide scale for both normal and cancerous tissues, serving as a reference for future studies and potential diagnostic applications of MALDI-IMS.

Abstract A010: ANO7 expression changes in prostate cancer progression — Association with an aggressive phenotype?

Abstract Introduction The expression level and single nucleotide polymorphisms of the prostate-specific gene ANO7 have been shown to correlate with prostate cancer (PrCa) outcome. Very little is known about ANO7, apart from its expression profile in tissues and that the protein is either a calcium-activated ion channel or a lipid scramblase. Our results suggest that ANO7 likely has a role in PrCa progression and that loss of ANO7 is associated with malignant PrCa. Materials and methods Sections of a prostate cancer formalin-fixed paraffin-embedded tissue microarray were used for ANO7 mRNA and protein detection. mRNA was detected with fluorescent in situ hybridization (FISH). Protein was detected with immunohistochemistry (IHC) using a polyclonal anti-ANO7 antibody (HPA035730). mRNA from fresh radical prostatectomy primary tumor samples were sequenced and subsequent gene expression levels were analyzed for co-expression with ANO7. The sample set consisted of a matched tumor and benign tissue sample (15+15) from each patient. Results The FISH images indicated explicitly that ANO7 mRNA is exclusively expressed in the luminal cells of the prostatic glandular epithelium. ANO7 mRNA is expressed in all cancerous lumina-forming glandular structures, but not in poorly-formed glands or sheets of cancer, in which ANO7 mRNA expression is very low or non-existent. Morphologically benign glands amidst cancerous tissue showed no expression, in contrast to tissue in which benign glands are predominating. The IHC showed a similar staining pattern to FISH.ANO7 co-expression gene set enrichment analysis resulted in 19 positively correlating and 77 negatively correlating pathways in KEGG (Kyoto Encyclopedia of Genes and Genomes) terms. Most of the positively correlating terms relate to metabolism, whereas among the negatively correlating terms, there were many signal transduction and cell growth and death-related pathways, such as Hippo, Ras, and p53 signaling pathways. Conclusions ANO7 mRNA and protein expression correlate spatially in the tissue, showing that the expression is specific to differentiated luminal cells and highly reduced in high-grade cancer. This notion is supported by the co-expressed gene-set enrichment analysis, which is showing a negative correlation of ANO7 with many pathways that are associated with aggressive PrCa. Mainly metabolism-related processes were positively correlated with ANO7, which could indicate that ANO7 is linked to the reprogramming of metabolism in the cells during cancer progression. Further research aims to identify the pathways in which ANO7 plays a significant role and to elucidate how the ANO7 protein functionally alters signaling related to PrCa progression. Citation Format: Olli Metsälä, Gudrun Wahlström, Pekka Taimen, Johanna Schleutker. ANO7 expression changes in prostate cancer progression — Association with an aggressive phenotype? [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A010.

Comprehensive FISH testing using FFPE tissue microarray of primary lymph node tissue identifies secondary cytogenetic abnormalities in Mantle Cell Lymphoma

IntroductionMantle Cell Lymphoma (MCL), is characterised by the reciprocal translocation t(11;14) resulting in CCND1-IGH gene fusion and subsequent upregulation of the CCND1 gene. Rearrangements of MYC and losses of CDKN2A and TP53 have been identified as biomarkers informing prognostic and potentially therapeutic information however these are not routinely assessed in MCL investigation. We aimed to identify additional cytogenetic changes using fluorescence in situ hybridisation (FISH) on formalin fixed paraffin embedded (FFPE) primary lymph node tissue microarrays in a cohort of 28 patients diagnosed with MCL between 2004 and 2019. FISH results were compared with corresponding immunohistochemistry (IHC) biomarkers to determine if IHC was a reliable screening tool to direct FISH testing. MethodFFPE lymph node tissue samples were constructed into tissue microarrays (TMA) which were stained with 7 immunohistochemical biomarkers: Cyclin D1, c-Myc, p16, ATM, p53, Bcl-6 and Bcl-2. The same TMAs were hybridised with FISH probes for the corresponding genes; CCND1-IGH, MYC, CDKN2A, ATM, TP53, BCL6 and BCL2. FISH and the corresponding IHC biomarkers were analysed to determine if secondary cytogenetic changes could be identified and if IHC could be used as a reliable, inexpensive predictor of FISH abnormalities to potentially direct FISH testing. ResultsCCND1-IGH fusion was detected in 27/28 (96%) of samples. Additional cytogenetic changes were identified by FISH in 15/28 (54%) of samples. Two additional abnormalities were detected in 2/28 (7%) samples. Cyclin D1 IHC overexpression was an excellent predictor of CCND1-IGH fusion. MYC and ATM IHC were useful screening tests to direct FISH testing and identified cases with poor prognostic features including blastoid change. IHC did not show clear concordance with FISH for other biomarkers. ConclusionFISH using FFPE primary lymph node tissue can detect secondary cytogenetic abnormalities in patients with MCL which are associated with an inferior prognosis. An expanded FISH panel including MYC, CDKN2A, TP53 and ATM should be considered in cases where anomalous IHC expression or is seen for these markers or if the patient appears to have the blastoid variant of the disease.

High mesothelin expression by immunohistochemistry predicts improved survival in pleural mesothelioma.

Mesothelin (MSLN) is a cancer-associated antigen that is overexpressed in malignancies such as mesothelioma, pancreatic and ovarian cancer. It is also a target for novel personalised therapies, including antibodies, antibody-drug conjugates and chimeric antigen receptor T cells. Immunohistochemistry may predict those who would best respond to anti-mesothelin therapies and guide decisions in therapeutic strategy. This study aimed to assess the intensity and distribution of MSLN immunostaining in mesothelioma, and to determine the prognostic value of MSLN expression by histochemical-score (H-score). The MN1 anti-MSLN antibody was used to stain a formalin-fixed paraffin-embedded tissue microarray of histologically confirmed mesothelioma from 75 consecutive patients who had undergone pleurectomy with or without decortication. MSLN positivity, the staining intensity, distribution of staining and H-score were evaluated. The correlation of H-score with prognosis was investigated. Sixty-six per cent of epithelioid tumours were MSLN-positive (with expression in > 5% tumour cells). Of MSLN-expressing epithelioid tumours, 70.4% had moderate (2+) or strong (3+) intensity MSLN immunostaining, although only 37% of samples had staining in ≥ 50% of tumour cells. In multivariate analysis, MSLN H-score as a continuous variable and an H-score ≥ 33 were independent predictors of improved survival (P = 0.04 and P < 0.001, respectively). MSLN expression was more heterogenous in epithelioid mesothelioma than reported previously. Therefore, it would be appropriate to perform an immunohistochemical assessment of MSLN expression to stratify and assess patient suitability for mesothelin-targeted personalised therapies, such as chimeric antigen receptor T cells.

Association of immunohistochemical profiles with histotypes in endometrial carcinomas

ObjectiveAlthough a large number of endometrial cancer patients are cured with surgery alone, there are significant numbers of patients with more aggressive variants of endometrial carcinoma for whom the prognosis remains poor. We investigated the effects of prevalence, histotypes, and immunohistochemical profiles on prognostic value in a hospital-based population. Materials and methodsA retrospective study of surgically resected primary endometrial carcinoma was included. Immunohistochemical stains were performed on formalin-fixed paraffin-embedded tissue microarray sections for β-Catenin, estrogen receptor (ER), progesterone receptor (PR), HER-2, MLH1, MSH2, MSH6, PMS2, and p53. ResultsLoss of mismatch repair expression was detected in 25.4% of samples (29/114, mean age 57 years) of the tumors. The following loss of expression was observed in patients: MLH1/PMS2 in 16.6% of patients, MSH6 in 7.0% of patients, MLH1 in 0.9% of patients, and MSH6/PMS2/MLH1 in 0.9% of patients. Immunohistochemistry of p53 was analyzed for 111 patients. A total of 13 patients (11.7%, mean age 64 years) had p53-abnormal expression (absent, cytoplasmic or diffuse strong positive patterns), and more than half (9/13, 69.2%) had endometrioid histotype. Abnormalities in p53 were significantly associated with histotype (p = 0.001), advanced tumor stage (p = 0.038), death of disease (p = 0.002), PR percentage (p = 0.002), and HER-2 expression (p = 0.018). Immunohistochemical nuclear localization of β-Catenin was detected in 7.1% of the cohort. The combination of p53 and nuclear β-Catenin expressions was not significantly predictive of disease-free or overall survival. ConclusionThe results of this study are useful for management of endometrial cancer in patients with DNA mismatch repair, abnormal p53 expression, or nuclear localization of β-Catenin.

In Situ N-Glycosylation Signatures of Epithelial Ovarian Cancer Tissue as Defined by MALDI Mass Spectrometry Imaging.

Simple SummaryAltered glycosylation of proteins was shown to be implicated in various steps of malignant transformation and tumor progression. For that reason, it is frequently referred to as the next hallmark of cancer. In epithelial ovarian cancer (EOC), a female gynecological malignancy of the highest mortality, N-glycosylation alterations were predominantly investigated at the level of serum glycoproteins and OC cell lines. By contrast, data on in situ N-glycosylation in OC tissue are still very limited, particularly with respect to terminal sialylation. This is despite the increasing number of studies supporting the role of sialylated N-glycans in tumor progression, angiogenesis, and metastasis. In this work, MALDI mass spectrometry imaging (MALDI-MSI) was implemented in combination with chemical sialic acid derivatization to determine tissue type-specific N-glycosylation of less common histotypes of EOC and non-malignant ovarian disease.The particularly high mortality of epithelial ovarian cancer (EOC) is in part linked to limited understanding of its molecular signatures. Although there are data available on in situ N-glycosylation in EOC tissue, previous studies focused primarily on neutral N-glycan species and, hence, still little is known regarding EOC tissue-specific sialylation. In this proof-of-concept study, we implemented MALDI mass spectrometry imaging (MALDI-MSI) in combination with sialic acid derivatization to simultaneously investigate neutral and sialylated N-glycans in formalin-fixed paraffin-embedded tissue microarray specimens of less common EOC histotypes and non-malignant borderline ovarian tumor (BOT). The applied protocol allowed detecting over 50 m/z species, many of which showed differential tissue distribution. Most importantly, it could be demonstrated that α2,6- and α2,3-sialylated N-glycans are enriched in tissue regions corresponding to tumor and adjacent tumor-stroma, respectively. Interestingly, analogous N-glycosylation patterns were observed in tissue cores of BOT, suggesting that regio-specific N-glycan distribution might occur already in non-malignant ovarian pathologies. All in all, our data provide proof that the combination of MALDI-MSI and sialic acid derivatization is suitable for delineating regio-specific N-glycan distribution in EOC and BOT tissues and might serve as a promising strategy for future glycosylation-based biomarker discovery studies.

Microtubule-Associated Protein 2 as a DHEA Binding Protein in Endometrial Cancer

Dehydroepiandrosterone (DHEA) is an androgen secreted by the adrenal glands, but its binding affinity for the androgen receptor is very low. DHEA is transformed into androstenedione by 3β-hydroxysteroid dehydrogenase (HSD) and then into testosterone by 17β-HSD type 5, or into estrone by aromatase. DHEA is also converted into androstenediol by 17β-HSD type 1. Therefore, DHEA is considered to play an important role as a precursor hormone for sex steroid hormones. We performed a search for a protein having an amino acid sequence homology to the DHEA binding site of 17β-HSD type 1, and found that microtubule-associated protein 2 (MAP2) binds to DHEA (Laurine E et al., J Biol Chem. 2003). MAP2 expression is necessary for neurite extension and cessation of cell division. MAP2 is known to suppress migration and invasion and affect the assembly, stabilization, and bundling of microtubules in melanoma cells, but the function of MAP2 in endometrial cancer has not been clarified. In this study, we investigated the expression of MAP2 and its association with DHEA in order to clarify the direct non-receptor action of DHEA in endometrial cancer. We employed frozen and formalin-fixed paraffin-embedded (FFPE) tissues of 35 endometrial cancer tissues (G1, n=12; G2, n=10; G3, n=9; Serous, n=4). Hormone concentrations were measured by liquid chromatograph-tandem mass spectrometer from the frozen sample, and immunohistochemistry of MAP2 was performed using FFPE tissues. We also examined MAP2 immunoreactivity using 59 normal endometrial tissues (proliferative phase, n=33; secretory phase, n=26) of FFPE tissue microarray slides. MAP2 immunoreactivity was found in the cytoplasm of endometrial cancer cells, and the MAP2-positive rate was significantly higher in type 1 (G1 and G2) than in type 2 (G2 and G3). The cell proliferation marker Ki-67 index was significantly lower in the MAP2-positive group. MAP2 was also detected in the glandular epithelial cells of the normal endometrium. The MAP2-positive rate was lower in the proliferative phase than in the secretory phase. Furthermore, the concentration of DHEA in the cancer tissue was significantly higher in the MAP2-positive group than in the MAP2-negative group. MAP2 is known to act on the stability of microtubules and is thought to be involved in the suppression of proliferation and infiltration in cancer cells. It was suggested that DHEA is involved in the stabilization of MAP2 and suppresses the progression of cancer in a hormone receptor-independent manner.

Abstract PS18-27: Integrated immuno-genomic analyses in early breast cancer: Results from the Scandinavian breast group 2004-1 (SBG-2004-1) randomized phase II trial

Abstract Background: Given the emerging role of immune-related biomarkers in breast cancer (BC), little is known about specific immune cell composition patterns and genomic alterations and how these could interact in early BC setting. The aim of this proof-of-principle study is to describe immuno-genomic correlates within the tumor microenvironment using newly developed techniques for multiplexed fluorescent immunohistochemistry (mfIHC, Mezheyeuski et al., J Pathol. 2018) and high-throughput low-input genome sequencing (CUTseq, Zhang et al., Nat Commun 2019). Methods: The randomized SBG-2004-1 phase II trial evaluated the feasibility of tailored and dose-dense epirubicin and cyclophosphamide followed by docetaxel (EC→T) as adjuvant chemotherapy for early BC, enrolling a total of 124 patients. CUTseq was applied to gDNA extracted from archival low-input formalin-fixed paraffin-embedded (FFPE) tissue as well as peripheral blood samples of BC patients. mfIHC was performed on FFPE tissue microarrays, allowing simultaneous detection of six immune markers (CD4, CD8, PD-L1, PD-1, FoxP3, CD68) and quantification in an automated and tissue-compartment manner (Vectra® Polaris™ automated quantitative pathology imaging system and inForm® software, Akoya Biosciences). Patient characteristics and 10-year follow-up data were also available. Associations of different immune cell patterns and DNA copy number alterations (CNAs) with clinicopathological parameters and survival outcomes were evaluated using standard statistical methods.Results: 69/124 (55.6%) and 82/124 (66.1%) FFPE samples were available for CNA profiling (blood gDNA for normalization, n=33) and mfIHC, respectively. MYC gene was the most commonly amplified cancer-associated gene (n=44, 63.8%), and other frequent alterations included ERBB2 (n=31, 44.9%) and PIK3CA (n=14, 20.3%) amplification as well as TP53 (n=29, 42%) and PTEN (n=16, 23.2%) deletion. CNA burden (i.e., the percentage of the genome either amplified or deleted) was significantly correlated with higher tumor grade (p=0.013) but was not prognostic for relapse-free survival. CD4+ T-cells were the most abundant immune cell subpopulation, followed by CD68+ macrophages. The immune checkpoint markers PD-L1 and PD-1 were mostly expressed in CD4+ T-cells in both tumor and stroma compartments, while stromal PD-1+CD8+ and PD-1+CD68+ cell subsets were also among the ones with the higher cell density. CD4+ T-cell density was significantly correlated with tumor size, whereas the different immune cell infiltration patterns were not found to be prognostic. Combined immuno-genomic analyses revealed that high CNA burden was inversely associated with intra-tumoral CD4+ (Spearman’s r=-0.33, p=0.01) and CD8+ infiltration (Spearman’s r=-0.37, p=0.004). Conclusions: The present study indicates the feasibility of CNA profiling by CUTseq and immuno-profiling by mfIHC in FFPE samples obtained from BC patients. Moreover, our data provide a link between genomic alterations and the immune landscape in early BC and set the basis for further application of CUTseq and mfIHC in larger patient cohorts. Citation Format: Ioannis Zerdes, Michele Simonetti, Alexios Matikas, Luuk Harbers, Ning Zhang, Dimitrios Salgkamis, Susanne Agartz, Markella Zacharouli, Pablo Moreno-Ruiz, Johan Hartman, Artur Mezheyeuski, Jonas Bergh, Nicola Crosetto, Theodoros Foukakis. Integrated immuno-genomic analyses in early breast cancer: Results from the Scandinavian breast group 2004-1 (SBG-2004-1) randomized phase II trial [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 PS18-27.

Abstract 2001: Biomarker discovery in immunotherapy-treated melanoma patients with imaging mass cytometry

Abstract Background: Understanding the complexity of the tumor microenvironment could allow identification of new biomarkers that predict response to immunotherapy in cancer patients and anticipate the toxicity associated with the treatment. Since the number of targets measurable by quantitative immunofluorescence (QIF) is limited, we applied Imaging Mass Cytometry (IMC), a metal-conjugated antibody-based high-plex technology, to generate a multidimensional analysis of the microenvironment. We interrogated a 25-antibody panel, including tumor and immune cell markers, in a cohort of melanoma patients treated with immune checkpoint blockers. Methods: We used a validated, optimized panel of 25 antibodies conjugated to unique metals, including a DNA intercalator conjugated with 191/193Ir, for detection using HyperionTM (Fluidigm). Digital image analysis was performed using a newly designed version of the AQUA software (Navigate BP) to measure marker intensity in molecularly defined masks or compartments as follows: all cells (DNA intercalator Ir191/193), tumor cells (HMB45/S100), stroma (tumor cells subtracted from all cells), T cells (CD3), B cells (CD20) and macrophages (CD68). Results were compared to QIF and Digital Spatial Profiling (DSP), a NanoString technology based on primary antibodies conjugated to indexing DNA oligos. We examined archived formalin-fixed paraffin-embedded (FFPE) samples from a metastatic melanoma cohort treated with immunotherapy (n=60) in tissue microarray format. Results: Immune markers such as CD3, CD4 and CD8 showed a high correlation between the three multiplexing methods: IMC, DSP and QIF (r=0.48-0.89, p&amp;lt;0.0001). Moreover, high CD3 and CD8 levels assessed by IMC were statistically significantly associated with favorable outcome, confirming results previously published on the same cohort (Toki et al, CCR, 2018). Multivariable analyses revealed significant associations of MHC-I, CSF1R, IRF1, LAG3, PD1, MHC-II and beta2-microglobulin (B2M) in tumor with progression-free survival (PFS) independent of age, sex, mutation, stage, treatment, and prior immune checkpoint blockade treatment. In stroma, high TIM3 and high PD-L2 also predicted response to immunotherapy. Furthermore, we tested for validation by QIF B2M, MHC-I and CSF1R, predictive markers identified by IMC. However, only B2M showed a statistically significant correlation with overall survival. Conclusion: Using IMC technology and digital image analysis based on pixel colocalization, we were able to evaluate simultaneously 25 markers on FFPE tissue microarray samples. There was a high concordance with QIF and DSP for immune markers such as CD3, CD4 and CD8. A series of potentially predictive biomarkers for immunotherapy in metastatic melanoma were identified. Citation Format: Sandra Martinez-Morilla, Franz Villarroel-Espindola, Pok Fai Wong, Harriet Kluger, Maria Toki, Thazin New Aung, Vasiliki Pelekanou, Brian Bourke-Martin, David L. Rimm. Biomarker discovery in immunotherapy-treated melanoma patients with imaging mass cytometry [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 2001.

A Machine-learning Approach for the Assessment of the Proliferative Compartment of Solid Tumors on Hematoxylin-Eosin-Stained Sections.

We introduce a machine learning-based analysis to predict the immunohistochemical (IHC) labeling index for the cell proliferation marker Ki67/MIB1 on cancer tissues based on morphometrical features extracted from hematoxylin and eosin (H&E)-stained formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples. We provided a proof-of-concept prediction of the Ki67/MIB1 IHC positivity of cancer cells through the definition and quantitation of single nuclear features. In the first instance, we set our digital framework on Ki67/MIB1-stained OSCC (oral squamous cell carcinoma) tissue sample whole slide images, using QuPath as a working platform and its integrated algorithms, and we built a classifier in order to distinguish tumor and stroma classes and, within them, Ki67-positive and Ki67-negative cells; then, we sorted the morphometric features of tumor cells related to their Ki67 IHC status. Among the evaluated features, nuclear hematoxylin mean optical density (NHMOD) presented as the best one to distinguish Ki67/MIB1 positive from negative cells. We confirmed our findings in a single-cell level analysis of H&E staining on Ki67-immunostained/H&E-decolored tissue samples. Finally, we tested our digital framework on a case series of oral squamous cell carcinomas (OSCC), arranged in tissue microarrays; we selected two consecutive sections of each OSCC FFPE TMA (tissue microarray) block, respectively stained with H&E and immuno-stained for Ki67/MIB1. We automatically detected tumor cells in H&E slides and generated a “false color map” (FCM) based on NHMOD through the QuPath measurements map tool. FCM nearly coincided with the actual immunohistochemical result, allowing the prediction of Ki67/MIB1 positive cells in a direct visual fashion. Our proposed approach provides the pathologist with a fast method of identifying the proliferating compartment of the tumor through a quantitative assessment of the nuclear features on H&E slides, readily appreciable by visual inspection. Although this technique needs to be fine-tuned and tested on larger series of tumors, the digital analysis approach appears to be a promising tool to quickly forecast the tumor’s proliferation fraction directly on routinely H&E-stained digital sections.

In situ Metabolite Mass Spectrometry Imaging: New Insights into the Adrenal Gland.

The adrenal gland integrates catecholamine-producing neuroendocrine cells and steroid-producing cells with mesenchymal origin in a structured manner under one capsule and is a key regulator for vital bioactivity. In addition to adrenal-specific disease, dysregulation of adrenal hormones is associated with systemic effects, leading to undesirable metabolic and cardiovascular consequences. Mass spectrometry imaging (MSI) technique can simultaneously measure a broad range of biomolecules, including metabolites and hormones, which has enabled the study of tissue metabolic and hormone alterations in adrenal and adrenal-related diseases. Furthermore, this technique coupled with labeled immunohistochemistry staining has enabled the study of the pathophysiological adaptation of the adrenal gland under normal and abnormal conditions at different molecular levels. This review discusses the recent applications of in situ MSI in the adrenal gland. For example, the combination of formalin-fixed paraffin-embedded tissue microarray and MSI to tissues from patient cohorts has facilitated the discovery of clinically relevant prognostic biomolecules and generated promising hypotheses for new sights into physiology and pathophysiology of adrenal gland. MSI also has enabled the discovery of clinically significant tissue molecular (i. e., biomarker) and pathway changes in adrenal disease, particularly in adrenal tumors. In addition, MSI has advanced the ability to optimally identify and detect adrenal gland specific molecules. Thus, as a novel analytical methodology, MSI has provided unprecedented capabilities for in situ tissue study.

Cytoplasmic CK1ε Protein Expression Is Correlated With Distant Metastasis and Survival in Patients With Melanoma.

Casein kinase 1 epsilon (CK1ε) is a member of the casein kinase 1 family, which includes highly conserved and ubiquitous serine/threonine protein kinases. Recent research has revealed that CK1ε plays an important role in a variety of human cancer types; however, its role in human melanoma remains unclear. The aim of this study was to elucidate the clinical role of CK1ε in patients with melanoma. Samples from 34 patients with melanoma were analyzed by immunohistochemical staining. Formalin-fixed paraffin-embedded tissue microarrays were also examined by two histopathologists to assess CK1ε protein expression in humans. Cytoplasmic CK1ε protein expression was significantly lower in tumor tissue than in normal tissue. Lack of cytoplasmic CK1ε protein was significantly correlated with distant metastasis (p=0.022) and poorer survival (p=0.030). However, Kaplan-Meier survival analysis revealed that elevated expression of cytoplasmic CK1ε protein was not significantly associated with the overall survival of patients with melanoma. Univariate and multivariate analyses demonstrated that lack of cytoplasmic CK1ε protein expression was related to distant metastasis (p<0.001 and p=0.004), showing that CK1ε was a prognostic factor. CK1ε protein expression might serve as a prognostic indicator in the treatment of patients with melanoma.

PRDX3 is associated with metastasis and poor survival in uveal melanoma

AimsUveal melanoma (UM) is the most common primary intraocular malignancy in adults, and 40% develop fatal metastatic disease. Overexpression of thioredoxin-dependent peroxidase reductase (PRDX3) has been implicated in several cancers,...

Protein Expression Analysis in Uterine Cervical Cancer for Potential Targets in Treatment.

Specific markers in lesions of the human uterine cervix cancer (UCC) are still needed for prognostic, diagnostic and/or therapeutic purposes. In this study we evaluated key molecules at protein level between normal epithelium, cervical intraepithelial neoplasia (CIN1-3) and invasive cancer of a group of molecules previously reported at mRNA level. For that purpose, human formalin-fixed paraffin embedded tissue microarrays (TMAs) were constructed containing 205 Mexican tissue core specimens. Immunohistochemistry and quantitative analysis of histological staining was performed against twenty-two distinct proteins for each core and the processing platform ImageJ. In the progression of the disease we found key statistical differences for the proteins SEL1, Notch3 and SOCS3. High expressions of SEL1L, Notch3 and SOCS3 have potential value to increase the prognostic of UCC in combination with markers such as p16INK4a. This study identified key drivers in cervical carcinogenesis that should be evaluated for the development of UCC therapies.

The role of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) in peripheral nerve sheath tumours.

STAT3 is a pro-oncogenic transcription factor that requires phosphorylation for transcriptional activation. The aim of this study was to evaluate the role of phosphorylated STAT3 (pSTAT3) expression in neurofibromas, schwannomas, and malignant peripheral nerve sheath tumours (MPNSTs). Twenty-six neurofibromas, 62 schwannomas and 39 MPNSTs from a formalin-fixed paraffin-embedded tissue microarray were examined. Immunohistochemical analysis was performed with an anti-pSTAT3 (Tyr705) antibody. Nuclear expression was reviewed for both intensity and percentage of tumoral labelling. Distributions of disease-specific overall survival (DSOS) and event-free survival (EFS) were estimated with the Kaplan-Meier method, and compared between two pSTAT3 groups by use of the log-rank test. MPNSTs had higher median tumoral labelling than neurofibromas (P = 0.0012) or schwannomas (P = 0.0008). Moderate to strong pSTAT3 expression (defined as at least moderate labelling in ≥50% of cells) was found more frequently in MPNSTs than in neurofibromas (P = 0.026). Among MPNSTs, pSTAT3 expression differed between primary, recurrent and metastatic disease (P = 0.063 with increased expression in recurrent and metastatic cases). pSTAT3 expression (at least moderate labelling in ≥10% of cells) in primary MPNSTs was associated with worse DSOS (P = 0.048) and trended towards being associated with worse EFS (P = 0.063). Paired specimens revealed no increase in pSTAT3 expression in the recurrences or metastases relative to the primary tumour, suggesting that pSTAT3 expression may be an early indicator of aggressive disease at disease onset. pSTAT3 is expressed in a higher proportion of MPNSTs than neurofibromas and schwannomas. Moderate to strong pSTAT3 expression in ≥10% of cells was found to be a negative prognostic factor for DSOS among primary MPNSTs, suggesting a role of pSTAT3 in the progression of these tumours.

MALDI IMS and Cancer Tissue Microarrays.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) technology creates a link between the molecular assessment of numerous molecules and the morphological information about their special distribution. The application of MALDI IMS on formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMAs) is suitable for large-scale discovery analyses. Data acquired from FFPE TMA cancer samples in current research are very promising, and applications for routine diagnostics are under development. With the current rapid advances in both technology and applications, MALDI IMS technology is expected to enter into routine diagnostics soon. This chapter is intended to be comprehensive with respect to all aspects and considerations for the application of MALDI IMS on FFPE cancer TMAs with in-depth notes on technical aspects.

Typing of colon and lung adenocarcinoma by high throughput imaging mass spectrometry

In advanced tumor stages, diagnosis is frequently made from metastatic tumor tissue. In some cases, the identification of the tumor of origin may be difficult by histology alone. In this setting, immunohistochemical and molecular biological methods are often required. In a subset of tumors definite diagnosis cannot be achieved. Thus, additional new diagnostic methods are required for precise tumor subtyping. Mass spectrometric methods are of special interest for the discrimination of different tumor types. We investigated whether it is possible to discern adenocarcinomas of colon and lung using high-throughput imaging mass spectrometry on formalin-fixed paraffin-embedded tissue microarrays. 101 primary adenocarcinoma of the colon and 91 primary adenocarcinoma of the lung were used to train a Linear Discriminant Analysis model. Results were validated on an independent set of 116 colonic and 75 lung adenocarcinomas. In the validation cohort 109 of 116 patients with colonic and 67 of 75 patients with lung adenocarcinomas were correctly classified.The ability to define proteomic profiles capable to discern different tumor types promises a valuable tool in cancer diagnostics and might complement current approaches. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Abstract 4229: Physical expansion of tissue microarrays for high-resolution imaging of normal and cancer samples with conventional microscopy

Abstract BACKGROUND Archival formalin fixed paraffin embedded (FFPE) tissues are an excellent and abundant source of human cancer and normal tissues, useful for cancer research and diagnosis. Moreover, FFPE tissue microarrays enable analysis of hundreds of samples from many different patients on the same slide. Super resolution microscopes have been used for cancer tissue imaging; however, these methods are expensive and require long recording times, limiting their usefulness for the cancer research community. Recently, a new approach (Expansion Microscopy) was developed to enable physical magnification and high resolution imaging of cell lines and fresh-frozen (and fixed) mouse brain with conventional microscopes (Chen F, Tillberg PW, Boyden ES, 2015, Science 347:543-548). In the current study, we developed expansion microscopy for expanding and imaging formalin fixed paraffin embedded normal and cancer human tissues, both in tissue microarrays and whole tissue slides. METHODS Expansion microscopy (ExM) physically magnifies tissue samples by embedding them in a dense swellable polymer, anchoring key biomolecules to the polymer mesh, and adding water to swell the polymer. ExM physically magnifies the brain tissue with nanoscale isotropy and a post-expansion measurement error of 2-4%. Proteins can also be visualized by a modified immunofluorescence assay (Chen F et al., Science, 2015). In the present study, we optimized the ExM chemistry, labeling, and imaging methodologies to enable ExM to be used in cancer research and diagnosis, for morphological and protein imaging and analysis of both tissue microarrays and whole tissue slides on a wide variety of human tissues. Nuclei were detected by DAPI, and protein markers, including those of stroma (vimentin) and epithelium (keratins), were detected using immunofluorescence. RESULTS and CONCLUSIONS We developed ExM protocols to enable expansion of human normal and cancer tissues ∼4.5x in linear dimension, with a post-expansion measurement error of 5% or less. We successfully expanded normal and cancer human FFPE tissue microarrays containing over eight different tissue origins, including breast, prostate, lung, colon, pancreas and ovary. We expanded fresh frozen tissue samples of normal and cancer tissues. Our method makes possible the use of ExM on routinely collected FFPE pathology samples, enabling super resolution optical investigation of morphology and protein expression/localization in tissue microarrays with conventional fluorescent microscopy. Future applications include both large-scale retrospective and prospective studies of carcinogenesis in clinical tissue samples. * Yongxin Zhao and Octavian Bucur contributed equally; # Edward Boyden (esb@media.mit.edu) and Andrew H. Beck are corresponding authors (abeck2@bidmc.harvard.edu); Citation Format: Octavian Bucur, Yongxin Zhao, Edward Boyden, Andrew H. Beck. Physical expansion of tissue microarrays for high-resolution imaging of normal and cancer samples with conventional microscopy. [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 4229.

Abstract 3451: Breast cancer risk factor associations by loss of E-cadherin tumor tissue expression: A pooled analysis of 5,896 cases in 12 studies from the Breast Cancer Association Consortium (BCAC)

Abstract Purpose: Expression of the tumor suppressor gene E-cadherin is diminished in lobular breast cancers and has been implicated in epithelial mesenchymal transition. We assessed risk factor associations for breast cancer stratified by low vs. high E-cadherin protein expression in a pooled analysis within the Breast Cancer Association Consortium (BCAC) studies. Methods: E-cadherin tumor tissue staining was performed centrally at the NCI on formalin-fixed paraffin-embedded tissue microarray (TMA) sections representing 6,010 breast cancer patients from 12 US and European BCAC studies. TMAs were digitally scanned and scored using the SlidePath Digital Image Hub (Leica Biosystems, Wetzlar, Germany). For 5,896 cancers with evaluable tumors, E-cadherin was visually scored as estimated percent of positive cells times stain intensity (0, 1+, 2+, 3+) (score range 0-300). E-cadherin low was defined as tumors with a score &amp;lt; 100. Risk factor associations for low vs. high E-cadherin expressing tumors were evaluated by logistic regression, adjusted for age and study site, and stratified by ER status and histologic subtype. To assess the consistency in results by study, meta-analyses were performed using the random-effects model. All statistical tests were two-sided. Results: E-cadherin low cancers comprised 20% of tumors and were associated with lobular histology, well/moderately differentiated cancers, &amp;gt; 2cm in size, and HER2-negative status (χ2, P &amp;lt; 0.003 for all factors). E-cadherin low status was associated with family history of breast cancer (FH) (OR = 1.32, 95% CI = 1.09-1.60, P-het = 0.005) and ever use of menopausal hormones (OR = 1.26, 95% CI = 1.02-1.56, P-het = 0.03). Study specific meta-plots showed consistent effects across studies for menopausal hormone therapy (I2 = 0.0%, p = 0.64); however, E-cadherin loss by FH did show evidence of heterogeneity by study (I2 = 54%, P = 0.03). Differences in E-cadherin expression remained significant for FH, and menopausal hormone use when further adjusted for ER (P &amp;lt; 0.05). We also found relationships with E-cadherin loss to vary by BMI, number of live births, age at first birth, and oral contraceptive use in stratified analysis by ER status and histologic subtype. Conclusion: This large pooled analysis shows that breast cancer risk factor associations may differ by E-cadherin expression independent of ER status, suggesting that it may represent a marker of etiologic heterogeneity. Citation Format: Hisani N. Horne, Hannah Oh, Mark E. Sherman, Maya Palakal, Stephen H. Hewitt, Marjanka Schmidt, Javier Benitez, Roger Milne, Hermann Brenner, Heli Nevanlinna, Arto Mannermaa, Georgia Chenevix-Trench, Fergus Couch, Peter Devilee, Diana Eccles, Maartje Hooning, Anthony J. Swerdlow, Nick Orr, Melissa A. Troester, Renata Cora, Paul D. Pharoah, Montserrat Garcia-Closas, Jonine D. Figueroa. Breast cancer risk factor associations by loss of E-cadherin tumor tissue expression: A pooled analysis of 5,896 cases in 12 studies from the Breast Cancer Association Consortium (BCAC). [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 3451.

Abstract 3110: MASH1 expression in high grade astrocytoma may demonstrate a potential model for prognosis that may lead to a molecular target for future therapy

Abstract Introduction: A large U.S. study showed that 25% of patients diagnosed with grade 3 astrocytoma survived for 5 years or more after diagnosis. Unfortunately, the outlook is even worse for patients diagnosed with GBM, as most patients live for less than a year. Achaete-scute complex homolog 1 (ASCL1, MASH1) is a proneural transcription factor that has an essential role in the formation of multiple neuronal lineages. Recent studies have shown that the upregulation of MASH1 and inhibition of the Notch signaling pathway characterize progressive astrocytoma. Proneural proteins have been shown to regulate stem-cell proliferation and differentiation. The convergence with oncogenic pathways combined with the overexpression in glioblastoma has been shown to correlate with poor clinical outcome and has identified these transcription factors as promising therapeutic targets. Recently, a MASH1 mouse monoclonal antibody has been developed. Most studies have shown MASH1 to be a specific marker of neuroendocrine carcinomas; however, only one immunohistochemical study on astrocytoma using a polyclonal antibody has been reported. This study examined MASH1 expression in grade 1-4 astrocytoma cases. Materials and Methods: Formalin-fixed paraffin-embedded tissue microarrays consisting of samples of normal brain and astrocytomas [grade 1 (n = 22), grade 2 (n = 34), grade 3 (n = 16) and glioblastoma (n = 35)] were evaluated by immunohistochemical analysis using a monoclonal MASH1 antibody. A value of &amp;gt;1% staining was scored as positive. Results: MASH1 stained 61.7% (66/107) of astrocytomas (grades 1-4). In grade 1 astrocytoma, MASH1 was expressed in 31.8% versus 85.3% in grade 2, 68.7% in grade 3 and 54.3% in grade 4 (p ∼ 0.0028). Conclusion: MASH1 is highly expressed in astrocytoma. There was a variable and progressive increase of MASH1 expression in grade 1 versus grade 2-4 astrocytoma; and thus, may demonstrate a potential model for prognosis that may lead to future target therapeutic strategies. Citation Format: David E. Tacha, Jillian Tyrrell, Margaret Lobo. MASH1 expression in high grade astrocytoma may demonstrate a potential model for prognosis that may lead to a molecular target for future therapy. [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 3110.