Paraffin-embedded Breast Cancer Tissues Research Articles

Amplified fluorescence imaging of HER2 dimerization on cancer cells by using a co-localization triggered DNA nanoassembly.

Convenient and sensitive detection of human epidermal growth factor receptor 2 (HER2) dimerization is highly desirable for molecule subtyping and guiding personalized HER2 targeted therapy of breast cancer. A colocalization-triggered DNA nanoassembly (CtDNA) strategy was developed for amplified imaging of HER2 dimerization. It exploits (a) the advantage of the specificity of aptamer proximity hybridization, and (b) the high sensitivity of hairpin-free nonlinear HCR. The mechanism of step-by-step hairpin-free nonlinear HCR for DNA dendritic nanoassembly was studied by native polyacrylamide gel electrophoresis, atomic force microscopy and fluorometry. The results revealed a high specificity, sensitivity, and excellent controllability of the DNA dendritic nanoassembly. The method was used to identify HER2 homodimers and HER2/HER3 heterodimers in various breast cancer cell lines using fluorescence microscopy. It was then extended to image and quantitatively evaluate HER2 homodimers in clinical formalin-fixed paraffin-embedded breast cancer tissue specimens. This revealed its remarkable accuracy and practicality for clinical diagnostics. Graphical abstract Schematic presentation of amplified imaging of human epidermal growth factor receptor 2 (HER2) dimerization on cancer cell surfaces by using a co-localization triggered DNA nanoassembly (CtDNA).

The composition of T cell infiltrates varies in primary invasive breast cancer of different molecular subtypes as well as according to tumor size and nodal status

T lymphocytes are the most numerous immune cells in tumor-associated infiltrates and include several subpopulations of either anticancer or pro-tumorigenic functions. However, the associations between levels of different T cell subsets and breast cancer molecular subtypes as well as other prognostic factors have not been fully established yet. We performed immunohistochemistry for CD8 (cytotoxic T cells (CTL)), FOXP3 (regulatory T cells (Tregs)), and GATA3 (Th2 cells) in 106 formalin-fixed paraffin-embedded invasive breast cancer tissue samples and analyzed both the numbers and percentages of investigated cells in tumor-associated infiltrates. We observed that triple-negative breast cancer (TNBC) and HER2+ non-luminal breast tumors were associated with more numerous CTLs and Tregs and a higher Treg/Th2 cell ratio as compared with luminal A subtype. A higher Treg percentage was related to a decreased hormone receptor expression, an increase in the Ki67 level, a greater tumor size of luminal tumors, and the presence of lymph node metastases. Moreover, differences in the composition of T cell infiltrates were associated with HER2 status and histologic grade and type, and a distinct immune pattern was observed in tumors of different phenotypes regarding pT stage and nodal status. The results of our work show the diversity of T cell infiltrates in primary invasive breast cancers of different phenotypes and suggest that progression of luminal or non-luminal tumors is related to distinct tumor-associated T cell composition.

Prognostic value of tumor cell DNA content determined by flow cytometry using formalin-fixed paraffin-embedded breast cancer tissues.

The use of formalin-fixed paraffin-embedded (FFPE) tumor tissues in flow cytometry (FCM)-based determination of tumor cell DNA content is more complicated than the use of fresh-frozen tissues. This study aimed to accurately measure tumor cell DNA content from FFPE tissues by separating tumor cells from stromal cells through FCM and investigating its prognostic impact. We separately measured the DNA contents of tumor cells and stromal cells by gating with pan-cytokeratin and vimentin (FCMC/V). We evaluated tumor cell DNA contents [DNA index (DI)] of 290 FFPE tumor tissues and classified them into low and high DI groups, using a cutoff DI value determined through an unbiased computational method. The distribution of DI was bimodal, and a cutoff value was determined at a DI of 1.26. The high-DI tumors were associated with aggressive phenotypes and had significantly worse distant recurrence-free intervals (DRFI) than low-DI tumors. Multivariate analysis revealed that lymph node metastasis, Ki67, and DI were independent factors affecting DRFI. Accordingly, patients with low-DI/low-Ki67 tumors had excellent outcomes compared with other tumor types. Multiploid tumors were associated with increased lymphocytic infiltration and aggressive phenotypes. The DI of FFPE tumors could be precisely determined through FCMC/V. A combination of DI and Ki67 analyses may be able to predict the prognoses of breast cancer patients with greater accuracy.

Abstract P3-06-17: Unlocking the transcriptomic potential of formalin-fixed paraffin embedded breast cancer tissues for high-throughput genomic analysis

Abstract Background: Transcriptomic analyses of clinical samples can help improve our understanding of disease aetiology, drug effectiveness, assign molecular subtypes and derive prognostic signatures for clinical decision-making. The success of early microarray studies relied heavily on sample quality and predominantly fresh frozen (FF) tissues to generate reliably robust data. The emergence of next-generation microarray and sequencing-based technologies from formalin-fixed paraffin-embedded (FFPE) tissues provides an opportunity to study archival clinical tissues with long-term follow-up. Here we assess 9 technologies, which vary in resolution, cost and RNA requirements, with matched FF and FFPE tissues from the same patient. Methods: Sequential tumour biopsies were taken pre-treatment and on-treatment (at 14-days and 3-months) from 11 postmenopausal patients with oestrogen receptor positive breast cancer treated with 3 months of neoadjuvant letrozole. Half of each sample was snap frozen in liquid nitrogen and half was FFPE, RNA was extracted from both. Transcriptomic analyses were performed using 9 technologies: Illumina Beadarray, Affymetrix U133A, Affymetrix Clariom S, NanoString nCounter, AmpliSeq Transcriptome, Lexogen QuantSeq and IonXpress RNAseq, Tempo-Seq BioSpyder and Qiagen UPX3'. Results: Success rates for generating robust expression profiles from FFPE tissues were 100% all except the Illumina BeadChip (22%) and AmpliSeq Transcriptome (83%) , which varied by the age of tissue. With the total number and position of probes/primers/counts varying widely between approaches, in total 7305 genes were represented across all of the whole-genome technologies tested. Clear batch effects were evident when comparing data from FF and FFPE tissues and when comparing between different technologies. Standard batch correction approaches such as XPN and ComBat minimised technical bias effect and increased the correlations between matched samples (FF and FFPE) to R>0.9, irrespective of the technology used. When analysed by multi-dimensional scaling following batch correction, samples clustered by treatment time-point. When ranked by expression of 60 proliferation genes, reported by us to change with letrozole treatment, samples ordered again by time-point, consistent with our previous findings, and paired samples clustered together. Conclusions: · Robust gene expression profiles can be reliably generated from FFPE tissues and are comparable to those derived from FF tissue using established transcriptomic approaches. · A range of new technologies are available for the study of FFPE tissues; these vary in cost, resolution and RNA requirements to fit the user's needs. · Gene expression data from biologically similar studies, generated using different technologies, can be reliably integrated for robust meta-analysis, subject to appropriate batch correction analysis. Citation Format: Turnbull AK, Selli C, Martinez-Perez C, Fernando A, Renshaw L, Keys J, Figueroa JD, He X, Tanioka M, Munro A, Murphy L, Fawkes A, Clark R, Coutts A, Perou CM, Carey LA, Dixon JM, Sims AH. Unlocking the transcriptomic potential of formalin-fixed paraffin embedded breast cancer tissues for high-throughput genomic analysis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-17.

Indoleamine 2,3-dioxygenase expression and overall survival in patients diagnosed with breast cancer in Pakistan.

BackgroundImmune dysfunction in breast cancer patients is well established. Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme that is linked with progression of cancer. IDO is overexpressed in triple-negative breast cancer (TNBC) cases.Materials and methodsWe conducted the first study to analyze IDO expression and overall survival in breast cancer cases in Pakistan. Expression of IDO, estrogen receptor (ER), progesterone receptor (PR), and human EGF receptor 2 (HER2) was evaluated by immunohistochemistry. Formalin-fixed paraffin-embedded breast cancer tissues of 100 (TNBC, n=49 and non-TNBC, n=51) patients were obtained from Shaukat Khanum Memorial Cancer Hospital and Research Centre. IDO expression was analyzed in association with clinicopathological features and overall survival. A total of 100 patients were classified based on the ordinal IDO score variables as low, medium, and high. In addition, overall mean age and SD of patients was 48.28±11.82.ResultsImmunohistochemical analysis showed that high IDO was observed in the TNBC patients (65.3%) compared to that in the non-TNBC patients (33.3%). Multivariable analyses showed that TNBC was an independent risk factor for high IDO expression. Overall survival was also significantly associated with IDO score.ConclusionOur study showed that IDO protein expression is higher in TNBC patients (P<0.01) and may suggest its role in disease pathogenesis. TNBC might be effectively treated with IDO inhibitors. Furthermore, high IDO expression is considerably associated with overall decreased patient survival. IDO might be utilized as a potential biomarker and immunotherapeutic target in breast cancer patients.

Amplicons in breast cancers analyzed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

Gene amplification is a common event in breast cancer, and identifies actual and potential targets of molecular therapy. The aim of the present study was to determine the amplification status of 22 genes that are reportedly frequently amplified in breast cancers. An archive of 322 formalin-fixed and paraffin-embedded invasive breast cancer tissues were screened by multiple ligation-dependent probe amplification (MLPA) and a total of 906 gene loci judged as 'gain' or 'amplified' was further confirmed to have been amplified based on fluorescence in situ hybridization (FISH). The results showed that 109 of 322 tumors (34%) displayed gene amplification of at least one of the 22 genes. The frequencies of the amplification of four regions containing known driver oncogenes were as follows: 8p11 (ZNF703, FGFR1, ADAM9, IKBKB), 8q24 (MYC), 11q13 (CCND1, C11ORF30), and 17q11-21 (CPD, MED1, ERBB2, CDC6, TOP2A, MAPT) exhibited amplification in 9.6%, 9.6%, 12.4%, and 12.1% of the tumors, respectively. In addition to homogeneously staining region- or double-minute chromosome-type amplifications, a centromere-associated-type amplification was found in nine tumors. Co-localization of the amplicon on 8p11 and the amplicon on 11q13 in single cells was found in 10 tumors, and in six of those tumors the two amplicons constituted single amplification units. Similarly, an amplicon consisting of ERBB2 and its flanking genes on 17q12-21 co-localized with an amplicon on 8p11 in 10 tumors and with the amplicon on 11q13 in five tumors. Thus, precise and feasible analysis of gene amplification status can be obtained using a combination of MLPA and FISH.

Stability of oestrogen and progesterone receptor antigenicity in formalin-fixed paraffin-embedded breast cancer tissue over time.

Use of archived formalin-fixed paraffin-embedded (FFPE) tissue is a standard method for evaluation of proposed prognostic and predictive tumour markers. However, little is known of the preservation of biomarker expression in old FFPE tumour blocks. We investigate the quality of immunohistochemical (IHC) oestrogen (ER) and progesterone receptor (PR) evaluation in FFPE tissue over time (1978-2000) using a large breast cancer tissue microarray (N=573) with access to receptor analyses in cytosol (CYT) at diagnosis, coexpression of other biomarkers and follow-up data. We found a good correlation between ER analysed with CYT at diagnosis and ER analysed with IHC in archived FFPE tissue from the same tumour. ER evaluation did not seem to be affected by tissue storage time. Nor was there any time-dependent difference in ERIHC correlation with other biomarkers (HER2, Ki67) or survival. Discordant cases were more often classified as ER-positive with IHC than with CYT. For PR, however, we found an increased correlation between methods in more recent time periods. This may possibly be explained by more reliable PRIHC results in newer samples, although other explanations may also contribute. Our results indicate stable ER expression in FFPE tissue archived for up to 40years.

RUNX3 Epigenetic Inactivation Is Associated With Estrogen Receptor Positive Breast Cancer.

The role of Runt-related transcription factor 3 ( RUNX3) gene in breast cancer remains not fully understood. We studied the correlation between RUNX3 gene promoter methylation and estrogen receptor (ER) expression status in breast cancer. Three breast cancer cell lines and 113 formalin-fixed, paraffin-embedded breast cancer tissue samples were analyzed for RUNX3 expression. Methylation-specific polymerase chain reaction was used to analyze RUNX3 methylation on the samples. Migration and invasion ability were evaluated in MCF7 cell line (RUNX3 methylated) treated with methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) to study the effect of RUNX3 methylation status. Our data showed that the expression of RUNX3 was high in MCF10A but not in MCF7 and SKBR3 cell lines, while the RUNX3 promoter showed hypermethylation in MCF7 but not in MCF10A and SKBR3. In tissues samples, Immunohistochemical (IHC) expression of RUNX3 protein was higher in ER-negative samples than in ER-positive cases, and it was negatively correlated with the methylation status of the RUNX3 gene promoter. Proliferation, migration, and invasion of MCF7 were suppressed when 5-Aza-CdR treated. Also, the hypermethylation status of RUNX3 gene promoter was reversed and RUNX3 expression was increased. In summary, our data suggest that hypermethylation of the RUNX3 gene promoter may play an important role in ER-positive breast tumor progression.

Clinicopathological significance of cancer stem cell markers CD44 and ALDH1 expression in breast cancer.

CD44 and aldehyde dehydrogenase 1 (ALDH1) has been reputed to be cancer stem cell (CSC) markers in breast cancer. Yet, the clinicopathologic and prognostic significance of these markers remain unclear. In this study, we have investigated the expression of these markers and their relation with conventional clinicopathologic tumor characteristic including molecular subtype. CD44 and ALDH1 expression were investigated by immunohistochemistry in a series of 157 formalin-fixed paraffin-embedded breast cancer tissues. Overall, CD44 and ALDH1 are, respectively, detected in 33% (52 of 157) and 7% (10 of 157) of breast cancer cases. We also observed that CD44 expression was associated with histological grade (p = 0.005). For ALDH1, we found that its expression is more frequent with elderly women (> 50 years, p = 0.03). The investigation of relationship between the stem cell phenotype and breast cancer molecular subtype, revealed that CD44 and ALDH1 expression was more frequent in basal-like tumors (p = 0.005). Among the two cancer stem cell markers tested, ALDH1 showed a strong association with the basal marker EGFR (p = 0.05). These findings suggest that CD44 and ALDH1 play a role in the clinical behavior in breast cancer and might be interesting biomarkers and therapeutic targets.

Mutation screening of the BRCA1 gene in sporadic breast cancer in the Central of Iran

The purpose of the work was to study mutation screening of BRCA1 in Iranian patients with sporadic breast cancer. We carried out a mutational analysis of BRCA1 gene in 101 breast cancer patients from a population in Central of Iran. The comparison of DNA of paraffin-embedded breast cancer tissue from patients was studied, and breast tissue from 30 unrelated normal women without cancer was selected as controls. The entire BRCA1 coding sequence was amplified by PCR with primers especially designed for comprehensive mutation screening by single-strand conformation polymorphism (SSCP) analysis. The PCR products revealing abnormal SSCP migration pattern were sequenced. Then, in silico investigation was performed to identify the effects of new mutations on stability and function of BRCA 1.A total of ten nucleotide alterations were observed in the breast cancer tissue DNA. Six cases of single nucleotide changes in BRCA1 were detected in the study without records in the BIC database consisted four polymorphism in exon 11 (1543 Del G, 1597C>T, 2246 Del T, 2612C>G), one missense mutation in exon 7 (490C>T), and one deletion mutation in exon 10 (743 Del C). No nucleotide alterations were detected in the controls. In addition, the results of in silico analysis indicated that three mutations including 2612C>G, 1543 Del G and 743 Del C were not recorded in the especial database. Furthermore, the results of molecular docking studies confirmed that 2612C>G could change physicochemical properties of BRCA1.The findings of the present study suggest that screened mutations may have an important role on the incidence of breast cancer in women.

A2A Receptors Are Highly Expressed in Young Patients with Luminal B and Triple Negative Breast Cancer Subtypes and Are Associated with a Higher Proliferate Index

Background and ObjectivesNumerous Molecules in the tumor microenvironment orchestrate the body's immune response against tumor cells thereby affecting tumor cell growth and proliferation. Adenosine is produced in the hypoxic tumor milieu and by stimulating its receptors (A2AR), plays an important immunosuppressive role that helps tumor cells evade the body s immune mechanisms. Furthermore, stimulation of these receptors also has a profound role in promoting the tumor vasculature and contributing to tumor aggressiveness. The present study aimed at investigating A2AR expression in breast cancer tissue and its association with different clinicopathological parameters of the tumor.MethodsThis study was conducted on 30 formalin‐fixed paraffin‐embedded (FFPE) human breast cancer tissues. Sections were immunohistochemically stained with of A2AR antibodies. Clinical and pathological data were retrospectively obtained from the patients' records in the archives of the pathology department at Alexandria University, Egypt.ResultsA2ARs were expressed not only on the tumor‐infiltrating immune cells (TILs) but were also found on the tumor cells themselves in 73.3% (n=22) of breast cancer tumors. Their expression on the tumor cells was significantly higher in younger age patients (&lt; 50 years) (88.2%) compared to older ones (54.5%) [p=0.044]. A2AR expression was also significantly associated with a Luminal B as well as triple‐negative tumors [p=0.028] compared to Luminal A and Her2‐enriched tumors. Patients expressing A2A receptors had a significantly higher expression of Progesterone receptors [P=0.031] compared to ER expression [P=0.098]. The majority of A2AR expressing tumors had a mitotic index score of 2 [p=0.013] as well as a significantly higher proliferative index (ki‐67 &gt;20%) [p=0.018]. No association was observed between A2AR expression and tumor size, type, grade, Lymph node status, the percentage of TILs or patient survival after 2 years of follow‐up.ConclusionOur study confirmed that A2AR were strongly expressed on breast cancer tumor cells in younger patients particularly those of luminal B and triple negative subtypes. Their expression was associated with a significantly higher proliferative index suggesting a possible role of these receptors in imparting a more aggressive phenotype and that targeting these receptors could have a potential role in improving the outcome of these patients.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased expression of biological markers as potential therapeutic targets in Saudi women with triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks the expression of hormone receptors and human epidermal growth factor receptor 2 (HER2). Although TNBC represents only 15% of all types of breast cancer, it accounts for a large number of metastatic cases and deaths. Because of the high metastatic rate and both local and systemic recurrence associated with TNBC, extensive research efforts are actively looking for target therapies to effectively treat this aggressive disease. Accordingly, this study has been initiated to investigate the differential expression of biological markers in TNBC and non-TNBC Saudi women that might be utilized as potential targeted therapy and/or predict the sensitivity to currently available therapeutic regimens. Two hundred formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues were selected and divided into 3 groups: benign breast tissues (20), TNBC tissues (80) and non-TNBC tissues (100). Expression of mRNA in FFPE tissues was analyzed using real-time polymerase chain reaction (RT-PCR) for the following genes: poly (ADP-ribose) polymerase 1 (PARP-1), topoisomerase 2A (TOPO-2A), vascular endothelial growth factor (VEGF), C-MYC, basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMP-2 and MMP-9), human epidermal growth factor 1 (HER1) and multidrug resistance (MDR) genes. In the TNBC group, expression of PARP-1, TOPO-2A, HER1, C-MYC, VEGF, bFGF and MMP-2 showed a highly significant increase compared to the non-TNBC group. The results of this study suggest that (1) TNBC patients will benefit more from TOPO-2A inhibitors as well as antiangiogenic and antimetastatic therapies; (2) inhibition of these target genes is emerging as one of the most exciting and promising targeted therapeutic strategies to treat TNBC in which the intended targets are DNA repair, tumor angiogenesis and metastasis.

Rate of Clinical Complete Response for 1 Year or More in Bone-Metastatic Breast Cancer after Comprehensive Treatments including Autologous Formalin-Fixed Tumor Vaccine.

Introduction No effective treatment has been developed for bone-metastatic breast cancer. We found 3 cases with clinical complete response (cCR) of the bone metastasis and longer overall survival of the retrospectively examined cohort treated comprehensively including autologous formalin-fixed tumor vaccine (AFTV). Patients and Methods AFTV was prepared individually for each patient from their own formalin-fixed and paraffin-embedded breast cancer tissues. Results Three patients maintained cCR status of the bone metastasis for 17 months or more. Rate of cCR for 1 year or more appeared to be 15% (3/20) after comprehensive treatments including AFTV. The median overall survival time (60.0 months) and the 3- to 8-year survival rates after diagnosis of bone metastasis were greater than those of historical control cohorts in Japan (1988–2002) and in the nationwide population-based cohort study of Denmark (1999–2007). Conclusion Bone-metastatic breast cancer may be curable after comprehensive treatments including AFTV, although larger scale clinical trial is required.

Quantifying Tertiary Lymphoid Structure-Associated Genes in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissues.

Tertiary lymphoid structures (TLS) have been detected in several types of human solid tumors. These structures are thought to regulate local adaptive immune responses that can promote or antagonize tumor progression. Despite positive prognostic values associated with a TLS presence in several studies, discrepancies still exist. TLS are structurally organized entities composed of varying numbers of multiple cell types making their assessment in tumor tissues, particularly biopsies, challenging. Immunohistochemical staining of TLS-related cell populations is the most frequently used method for identifying and scoring them; however, TLS-related gene expression has also been explored. The protocols described are detailed to allow the user to quantify TLS-related gene expression on formalin-fixed paraffin-embedded human breast tumor tissues.

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Association of Epstein - Barr virus and breast cancer in Eritrea

BackgroundThe oncogenic potential of Epstein-Barr virus (EBV) in breast cancer is being increasingly recognized. Despite some controversies regarding such role, new evidence is suggesting a culpability of EBV in breast cancer, particularly in Africa where the virus has been originally associated with causation of several solid and hematological malignancies. One example is a report from Sudan implicating EBV as a prime etiologic agent for an aggressive type of breast cancer, where nearly 100% of tumor tissues were shown to carry viral signatures. To get a broader view on such association, other nearby countries should be investigated. The present study aims to determine the prevalence and possible associations of the virus in Eritrean breast cancer patients.MethodsDetection of EBV genome using primers that target Epstein Barr Encoded RNA (EBER) gene and Latent Membrane Protein-1 (LMP-1) gene sequences was performed by polymerase chain reaction (PCR) on DNA samples extracted from 144 formalin fixed paraffin embedded breast cancer tissues and 63 non-cancerous breast tissue as control group. A subset of PCR positive samples was evaluated for EBER gene expression by in situ hybridization (ISH). Expression of Latent Membrane Protein-2a (LMP2a) was also assessed by immunohistochemistry in a subset of 45 samples.ResultsBased on PCR results, EBV genome signals were detected in a total of 40 samples (27.77%) as compared to controls (p-value = 0. 0031) with a higher sensitivity when using the EBER primers. Five out of the 14 samples stained by EBER-ISH 35.71% were positive for the virus indicating the presence of the viral genome within the tumor cells. Of those stained for IHC 7 (15.55%) were positive for LMP2a showing low viral protein frequency.ConclusionsBased on these findings it can be concluded that EBV in Eritrea is associated with a smaller subset of tumors, unlike neighboring Sudan, thus pointing to possible differences in population predisposition and diseases epidemiology.

Elevated Hu-Antigen Receptor (HuR) Expression is Associated with Tumor Aggressiveness and Poor Prognosis but not with COX-2 Expression in Invasive Breast Carcinoma Patients.

Hu-antigen R (HuR), a RNA-binding protein, is considered to play a crucial role in tumor development and progression by stabilizing or regulating a group of cellular mRNAs of cancer-related genes, such as cyclooxygenase-2 (COX-2). The present study aimed to evaluate the clinical significance of HuR and COX-2 expression in invasive breast carcinoma. HuR and COX-2 protein expression was assessed immunohistochemically on paraffin-embedded breast cancer tissue sections obtained from 121 patients and was statistically analyzed with clinicopathological parameters, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), as well as with tumor cells' proliferative capacity and overall and disease-free patients' survival. High HuR expression was positively associated with larger tumor size and advanced disease stage (p=0.0234 and p=0.0361, respectively), being more frequently observed in ER negative cases (p=0.0208). High COX-2 expression was negatively associated with histological (p<0.0001) and nuclear (p=0.0033) grade and tumor cells' proliferative rate (p=0.0015), being more frequently observed in luminal-A compared to other molecular subtypes (p=0.0221). High HuR expression was associated with poor overall and disease-free patients' survival at both univariate (log-rank test, p=0.0092 and p=0.0004, respectively) and multivariate (Cox-regression analysis, p=0.0223 and p=0.0004, respectively) level. On the other hand, high COX-2 expression was associated with favorable overall and disease-free patients' survival merely at univariate level (log-rank test, p=0.0389 and p=0.0154, respectively). HuR expression was not associated with COX-2 expression (Spearman R=0.1489, p=0.1032). The present data support evidence that HuR is associated with tumor aggressiveness and poor prognosis in breast carcinoma, reinforcing its potential as promising therapeutic target in this type of neoplasia.

Overexpression of p53 gene has an important role in induction and progression of breast carcinoma

Breast cancer is the most common cancer in women, accounting for 23% of all female cancers around the globe. breast cancer molecular disease occurs due to the alteration in the genes that control cell growth and proliferation , such as HRE2/neu, c-MYC, K-RAS, RB, P53,PTEN,BRCA1 and BRCA2. The p53 tumor suppressor gene is located on chromosome 17p13, consists of 11 exons and encodes a53-kDa nuclear phosphoprotein, that has a very important function in many cellular processes, such as cell-cycle control, DNA repair , apoptosis and gene transcription. p53 is the most common mutated gene in human cancers , including breast. The aim of this study was to study the correlation between the overexpression of p53 and clinicopathological parameters in breast cancer patient. The current study included 85 sample of Paraffin-embedded breast cancer tissues, which were analyzed for p53 overexpression by immunohistochemistry. We also studies correlation between p53 overexpression and clinicopathological parameters. &#x0D; The results have clarified that 64.7% (55 out of 85) of cases of breast cancer had high expression of P53 immunostaining in their histological sections ,while 35.3% of cases were negative for p53 expression. The p53 expression was significantly correlated high grade and stage as well as age group less than 50 years ,and there was no significant correlation between p53 expression and gender of patient, lymph node status, tumor size, tumor site and histological types.&#x0D; Conclusion: These results demonstrated the overexpression of p53 in more than half of breast cancer cases and that overexpression of p53 was well correlated with grade and stage as well as young age group less than 50 years breast cancer patients.

Abstract 3833: Systematic evaluation of transcriptome sequencing shows comparable profiles for an exome-capture method for formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues and the standard poly-A method for matched fresh frozen (FF) tissues

Abstract Background: Tissue samples are routinely preserved as FFPE blocks. This abundant sample reservoir holds promises in clinical omics for disease diagnosis and precision cancer medicine. However, due to extensive cross-linking, nucleic acids are not efficiently extracted from FFPE samples and are heavily degraded, posing challenges for molecular assays including next generation sequencing. Our study aims to identify the RNA sequencing (RNA-seq) library prep method for FFPE samples that provides comparable data to the established poly-A enrichment method for FF tissue. Methods: RNA-seq data were collected on matched FF and FFPE samples from triple negative breast cancer (TNBC; n=7) and normal breast tissues (n=2). Tissues were sourced from a commercial vendor. Three library preparation protocols from Illumina were tested: poly(A) enrichment protocol (Poly-A; FF only), ribosomal RNA deletion protocol (Ribo-Zero Gold), and exome-capture based protocol (RNA-Access). Data were compared on absolute and differential gene expression, as well as molecular subtyping for breast cancer (PAM50; Parker 2009). Results: Both absolute expression values and log fold changes between tumor and normal samples correlate strongly across different tissue preservation and library preparation methods. Among tested methods, RNA-Access had the highest yield of mappable reads on coding region and provided uniform transcript coverage with less rRNA contamination. Regardless of the protocol, all TNBC samples were classified as PAM50 basal-like subtype. As expected, FF with either Ribo-Zero or RNA-Access had the strongest correlation with FF PolyA on the distance to subtype centroids (r=0.99). For FFPE methods, RNA-Access (r=0.94) had the strongest correlation with FF PolyA, followed by Ribo-Zero (r=0.85). Principal component analysis showed that samples were primarily segregated by technical differences (library preparation or tissue preservation method) rather than biological differences (tumor/normal status or patient identification), suggesting strong technical bias in the data. Gene families and biological processes underlying the differentially expressed genes due to technical variations included ribosomal, histone and metabolic pathways proteins. The aforementioned technical bias can be largely eliminated by a batch effect adjustment method (COMBAT; Johnson 2007). Conclusion: As expected, RNA-seq data is primarily impacted by technical differences. RNA-Access based transcriptome sequencing on breast FFPE samples generated highly concordant gene expression profiles when compared to data derived from FF samples. Our study supports the use of RNA-Access based transcriptome sequencing on FFPE samples when FF samples are not available. Citation Format: Xi Zhao, Marianna Zavodovskaya, Luting Zhuo, Kevin Kwei, Xin Guo, Zhaoshi Jiang, Scott D. Patterson, Carrie B. Brachmann. Systematic evaluation of transcriptome sequencing shows comparable profiles for an exome-capture method for formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues and the standard poly-A method for matched fresh frozen (FF) tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3833. doi:10.1158/1538-7445.AM2017-3833