Fresh Frozen Breast Cancer Tissue Research Articles

Exploring the presence of bovine leukemia virus among breast cancer tumors in a rural state.

The bovine leukemia virus (BLV) is a deltaretrovirus that causes malignant lymphoma and lymphosarcomas in cattle globally and has high prevalence among large scale U.S. dairy herds. Associations between presence of BLV DNA in human mammary tissue and human breast cancer incidence have been reported. We sought to estimate the prevalence of BLV DNA in breast cancer tissue samples in a rural state with an active dairy industry. We purified genomic DNA from 56 fresh-frozen breast cancer tissue samples (51 tumor samples, 5 samples representing adjacent normal breast tissue) banked between 2016 and 2019. Using nested PCR assays, multiple BLV tax sequence primers and primers for the long terminal repeat (LTR) were used to detect BLV DNA in tissue samples and known positive control samples, including the permanently infected fetal lamb kidney cell line (FLK-BLV) and blood from BLV positive cattle. The median age of patients from which samples were obtained at the time of treatment was 60 (40-93) and all were female. Ninety percent of patients had invasive ductal carcinoma. The majority were poorly differentiated (60%). On PCR assay, none of the tumor samples tested positive for BLV DNA, despite having consistent signals in positive controls. We did not find BLV DNA in fresh-frozen breast cancer tumors from patients presenting to a hospital in Vermont. Our findings suggest a low prevalence of BLV in our patient population and a need to reevaluate the association between BLV and human breast cancer.

Abstract P6-14-17: Endomucins and their expression in breast cancer and the cellular and therapeutic impact

Abstract Introduction. Endomucin-1 and endomucin-2 are two proteins structurally related, yet with low homology. The membrane bound sialoglycoproteins appear to play a key role in interfering with the formation of focal adhesion complexes (FAC) and matrix adhesiveness of cells, by mechanisms independent of the MUC1 repeat, which the endomucins do not possess. Endomucins are thought to be expressed at high levels in endothelium and haematopietic cell lineages, although the levels in mammary tissues also seem high. Despite the seeming importance of endomucins in the adhesion and migration of cells, including cancer cells, the clinical value of endomucin in clinical cancer, including breast cancer, is largely unknown. The present study examined the expression profile of endomucins, together with the focal adhesion kinase FAK, in breast cancer and aimed to explore the cellular impact of endomucin on cancer cells. Methods. Human breast cancer cells MCF-7 and MDA MB-231 and a range of other cell types were used. An endomucin overexpression cell model was created and subsequently used to evaluate the function of the cells. The expression profile of the endomucin-1 and endomucin-2 transcripts and FAK, in an existing fresh frozen breast cancer tissue cohort, were quantified. Results. High levels of endomucins, particularly endomucin-1, are good indicators for the overall survival of the patient, p=0.021 for endomucin-1 and p=0.15 for endomucin-2. When expression levels of FAK were integrated into the survival analysis model, patients with high levels of both endomucins and low levels of FAK had the most favourable outcome, compared with those with most unfavourable outcome who had low level of endomucin and high FAK (survival during the follow up period respectively at 100% and 54%, p=0.013, Harzoud Ratio 0.298). Together with the Nottingham Prognostic Index, which independently predicts a poor outcome (p=0.009, HR=7.6), the integrated expression profile of endomucin/FAK represents an independent prognostic indicator for favourable overall survival (p=0.003, HR=0.13), and indeed for a favourable disease free survival (p=0.008, HR=0.17). Mammary tissues, and indeed breast cancer cell lines, expressed high levels of endomucin-2 transcripts and low levels of endomucin-1 transcripts. High levels of endomucin-2 were also seen in fibroblasts and vascular endothelial cells. We created a breast cancer cell submodel with MCF-7, by overexpressing endomucin. It was shown that although endomucin over-expression had some marginal impact on the adhesiveness of breast cancer cells, the over-expression however, had significant impact on cells’ sensitivity to FAK inhibitor, with a markedly reduced adhesiveness to matrix (p< 0.001 control versus endomucin overexpression cells). Discussion. Endomucins have a reduced expression in breast cancers and the reduction, together with low levels of focal adhesion kinase, facilitate a favourable outcome for the patients. Together with the findings of in vitro cell models, it would suggest that the expression profile of endomucins and FAK may be a good indicator, not only for evaluating clinical outcomes, but also for choice of target therapies. Citation Format: Amber Xinyu Li, Tracey A. Martin, Lin Ye, Andrew J. Sanders, Fiona Ruge, Jane Lane, Eleri Davies, Wen G. Jiang. Endomucins and their expression in breast cancer and the cellular and therapeutic impact [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-17.

Abstract P2-11-33: Striatin and its interactive proteins Protein Phosphatase-2 (PP2) and PP2 regulatory elements in breast cancer

Abstract Introduction. The striatin protein family are calmodulin binding scaffolding proteins. Striatins have been involved in cell signalling and recently found also acting as cell adhesion molecules and adhesion regulators in adherens junctions and tight junctions. Straitins, particularly striatin-2 forms a recently discovered protein complex, STRIPAK, with other complex members including Protein Phosphatase-2 (PP2), STRIP, SIKE1 and certain PP2 regulatory proteins. The STRIPAK regulates multiple cell functions including gene transcription, endo- and exocytosis and cell adhesion, and several other important cell functions in cancer development and progression. The constitute members of the striatin protein complex may thus have important clinical bearings. In the present study, we investigated the expression profile of the striatin family members and key members of their interacting proteins, and discerned the relationship between the expression and disease progression and clinical outcome. Methods. Using an established fresh frozen breast cancer tissues cohort that included both normal mammary tissues and cancer tissues, we quantitatively evaluated the transcript expression of striatin-1 (STRN), striatin-3 (STRN3), straitin-4 (STRN4), its key regulator calmodulin (CALM) and the protein complex regulators protein phosphatase-2A (PPP2A), -2B (PPP2B) and the PPP2 regulatory elements PPP2R4 and PPP2R1A. The expression of each molecule was assessed against the clinical, pathological and prognostic factors of the patients. The integrated pattern of the complex members were also tested against the clinical outcome. Results. All three striatin members were expressed at good levels in mammary tissues and cancer tissues. STRN had little significant value against clinical and pathological factors. STRN3 and STRN4 were seen at high levels in tumours of high grade, with node positivity and with breast cancer related incidence. It was high level expressions of STRN3, STRN4 and CALM that were respectively associated with shorter overall survival (OS) of the patients and together they formed a poor prognostic indicator (p=0.034, HR=1.7). STRN had little impact on clinical outcomes. In a clear contrast, high levels of PPP2A, PPP2B and PPPR1A, but not PPP2R4, were seen in patients with significantly longer OS and together form a favourable prognostic indicator (p=0.034, HR=0.685). Integration of both STRN group and PPP2 group indicators constitutes a highly significant prognostic indicator for OS (p< 0.00001, HR=2.1 (95%CI 1.36-3.07)) and DFS (p=0.003, HR=1.402 (95%CI 1.12-1.75)). The predictive value of the integrated profile is independent of other clinical, pathological and hormone receptor status in multivariate analyses with OS (p< 0.0001, HR=3.861) and for DFS (p< 0.001, HR=2.055 (95%CI 1.36-3.07)). The same value stands when applied for the subtypes including triple negative breast cancers. Discussion. The STRIPAK complexes including Striatins play important regulatory roles in various cell functions and cancer development. Consistently, we found that high level expressions of STRN3, STRN4 and CALM, as a poor prognostic indicator were associated with shorter overall survival (OS) of the patients, while high levels of PPP2A, PPP2B and PPPR1A, as a favourable prognostic indicator, were seen in patients with significantly longer OS. Combination of both STRN group and PPP2 group indicators constitutes a greater significant prognostic indicator for OS and DFS. Future studies should focus on investigating the exact roles of the STRIPAK in cancer development and progression. Citation Format: Amber Xinyu Li, Andrew J. Sanders, Tracey A. Martin, Lin Ye, Fiona Ruge, QingPing Dou, Eleri Davies, Wen G. Jiang. Striatin and its interactive proteins Protein Phosphatase-2 (PP2) and PP2 regulatory elements in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-33.

PRAF2 is an oncogene acting to promote the proliferation and invasion of breast cancer cells.

Prenylated rab acceptor 1 domain family member 2 (PRAF2) acts as an oncogene and is closely related to the occurrence and development of various tumors. The present study aimed to clarify the functional relevance of PRAF2 in the biological behaviors of breast cancer by determining the expression of PRAF2 in breast cancer tissues and the corresponding adjacent tissues. The gene phenotypes of PRAF2 in patients with breast cancer in The Cancer Genome Atlas database were predicted using a cancer data online analysis website: The University of Alabama at Birmingham Cancer Data Analaysis Portal (UALCAN). The mRNA and protein expression of PRAF2 was further examined in 37 pairs of fresh frozen breast cancer tissues and adjacent non-tumor tissues by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. High expression of PRAF2 was verified by RT-qPCR in the breast cancer cell line, MCF-7, and small interfering RNA (siRNA) technology was used to silence PRAF2. In the in vitro cell functional experiment, three groups were used: Negative control (NC) group, siRNA-NC group and siRNA-PRAF2 group. Cell Counting Kit-8 (CCK-8) and colony formation assays were conducted to analyze the effect of downregulation of PRAF2 on the proliferation of breast cancer cells. Transwell invasion and cell scratch assays were performed to examine the effect of downregulation of PRAF2 on the invasion and migration of breast cancer cells. UALCAN analysis results indicated that PRAF2 expression was upregulated in breast cancer compared with normal tissue samples (P<0.001). High expression of PRAF2 in breast cancer was associated with TNM stage and regional lymph node metastasis. RT-qPCR results showed increased mRNA expression of PRAF2 in clinical tissue samples from 37 patients with breast cancer, compared with normal adjacent tissues (P<0.001). Protein expression of PRAF2 was also shown to be higher in the breast cancer MCF-7 cells than in the MDA-MB-231 cells. Western blotting analysis combined with ImageJ software quantification showed that the relative expression of PRAF2 protein was significantly higher in clinical tissue samples from 37 patients with breast cancer (1.9750±0.0103) than that in normal adjacent tissues (0.9818±0.0140) (P<0.001). Western blotting analysis results indicated that transfection with siRNA PRAF2 in MCF-7 cells decreased PRAF2 expression (P<0.001). The results of CCK-8 and colony formation assays revealed that downregulation of PRAF2 expression suppressed the proliferation of MCF-7 cells (P<0.05 and P<0.001, respectively). In addition, Transwell invasion and cell scratch assay results showed that downregulation of PRAF2 expression in MCF-7 cells repressed invasion and migration of cancer cells (P<0.001). Overall, PRAF2 expression was significantly higher in breast cancer tissues than normal adjacent tissues, and was closely related to TNM stage and regional lymph node metastasis in breast cancer. PRAF2 was found to act as an oncogene that is able to promote breast cancer cell proliferation and invasion. Thus, PRAF2 may be a potential prognostic factor in patients with breast cancer and a potential target for the treatment of breast cancer metastasis.

Effect of seasonal variation in ambient temperature on RNA quality of breast cancer tissue in a remote biobank setting

Studies involving oncology especially diagnosis, prognosis and therapeutic monitoring are increasingly relying on molecular analyses. These analyses require high quality biomolecules to get accurate and precise results and this requires among others, monitoring for pre-analytical variables. The purpose of our study was to validate the SOPs of the newly established Egyptian National Cancer Institute (ENCI) biobank. We used a panel of 91 fresh frozen breast cancer tissue samples and their matched normal tissues and have investigated the overall quality (integrity and yield) of RNA extracted from fresh frozen breast tumor tissues and matched normal breast tissues. We investigated the effect of several factors including seasonal temperature variation, cold ischemia time, transportation method, and RNA extraction method. The RNA yield and quality were significantly increased with tumor samples collected in winter, transported on wet ice and using an automated RNA extraction platform. No significant effect was observed due to increased cold ischemia time >30 min. The effect of delay in time to cryopreservation on RNA degradation in fresh tissue samples may vary according to the type of tissue, temperature during tissue collection and transportation, and the use of stabilizing agents as RNA later.

Progression-specific genes identified in microdissected formalin-fixed and paraffin-embedded tissue containing matched ductal carcinoma in situ and invasive ductal breast cancers

BackgroundThe transition from ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) is an important step during breast carcinogenesis. Understanding its molecular changes may help to identify high-risk DCIS that progress to IBC. Here, we describe a transcriptomic profiling analysis of matched formalin-fixed and paraffin-embedded (FFPE) DCIS and IBC components of individual breast tumours, containing both tumour compartments. The study was performed to validate progression-associated transcripts detected in an earlier gene profiling project using fresh frozen breast cancer tissue. In addition, FFPE tissues from patients with pure DCIS (pDCIS) were analysed to identify candidate transcripts characterizing DCIS with a high or low risk of progressing to IBC.MethodsFifteen laser microdissected pairs of DCIS and IBC were profiled by Illumina DASL technology and used for expression validation by qPCR. Differential expression was independently validated using further 25 laser microdissected DCIS/IBC sample pairs. Additionally, laser microdissected epithelial cells from 31 pDCIS were investigated for expression of candidate transcripts using qPCR.ResultsMultiple statistical calculation methods revealed 1784 mRNAs which are differentially expressed between DCIS and IBC (P < 0.05), of which 124 have also been identified in the gene profiling project using fresh frozen breast cancer tissue. Nine mRNAs that had been selected from the gene list obtained using fresh frozen tissues by applying pathway and network analysis (MMP11, GREM1, PLEKHC1, SULF1, THBS2, CSPG2, COL10A1, COL11A1, KRT14) were investigated in tissues from the same 15 microdissected specimens and the 25 independent tissue samples by qPCR. All selected transcripts were also detected in tumour cells from pDCIS. Expression of MMP11 and COL10A1 increased significantly from pDCIS to DCIS of DCIS/IBC mixed tumours.ConclusionWe confirm differential expression of progression-associated transcripts in FFPE breast cancer samples which might mediate the transition from DCIS to IBC. MMP11 and COL10A1 may characterize pure DCIS with a high risk developing IDC.

Is HPV virus associated with human breast cancer? Time to re-examine the postulate. Egyptian Study

Background: There is increasing evidence that high-risk human papilloma virus (HPV) is involved in cancers in addition to cervical cancer. Infectious agents are thought to be responsible for approximately 16% of cancers worldwide, however there are mixed reports in the literature regarding the prevalence and potential pathogenicity of viruses in breast cancer. Methods : We screened 30 fresh frozen breast cancer tissue specimens collected immediately postoperative from patients in Ain-Shams University Hospital for the presence of human papilloma virus (HPV) DNA by PCR and Koilocytic changes in the breast cancer tissue. Results: Overall prevalence of HPV in malignant breast tissue was 16.7% In addition, we found that the oncogenic characteristics of HPV associated breast cancer are very similar to HPV-associated cervical cancer. Specifically, that putative koilocytes are present in some HPV associated breast cancers. Conclusion : The above observations indicate a likely causal role for high-risk HPV in human breast cancer and offer the possible role of HPV in breast cancer prognosis. DOI: 10.21276/APALM.2017.1143

Abstract P1-02-01: Genomic analysis of single cells isolated by a pulse laser retrieval system

Abstract Background: Isolating tumor cells of interest and harvesting histologically pure samples is important for genomic studies. Laser capture microdissection (LCM) is an established method to obtain such purified cell populations for various applications including DNA, gene expression, and single cell analyses. However, LCM possesses problems such as limited optical resolution, cell fragmentation from dissection, and adherence of adjacent tissue to the cells which interrupts with single cell isolation from tissue sections. To overcome these obstacles, we developed a high-throughput pulse laser retrieval system which uses a wavelength that minimizes damage to the cellular content and is processed with a sacrificial layer that provides applicable optical resolution. The aim of this study was to evaluate the performance of the pulse laser retrieval system to provide appropriate samples for genomic analysis using breast cancer tissue. Methods: An indium tin oxide (ITO) coated glass slide was prepared using fresh frozen breast cancer tissue sections of 4㎛ thickness and stained by hematoxylin and eosin. The slide was mounted on the cell isolation machine and imaging was performed with a charge-coupled device camera using a 20× lens. Following identification of the target cells by a pathologist, nano-second pulsed laser (wavelength= 1064nm) was irradiated on the target. Isolated cells were collected in a polymerase chain reaction tube and whole genome amplification (WGA) was carried out using Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Amplified genomic DNA was fragmented and Illumina sequencing libraries were constructed. Sequencing was carried out to generate data with 0.1∼0.2× depth throughout the whole genome for each sample. Copy number variation (CNV) was analyzed by the Variable binning algorithm. Results: Whole genome amplification was performed using bulk tissue and 10 captured single cells from the same specimen. No difference in amplification coverage was observed between the two samples. A CNV analysis of captured single cells revealed similar CNV profiles with those in a matched bulk tumor. Whole exome sequencing (WES) of captured single cells yielded a variant frequency of 15% at a read depth of 15× and 50M base coverage, compared to 0% at 100× and 50M for WES using bulk tumor and 0.5% at 1200× and 100K for targeted sequencing using bulk tumor. Laser capture was performed for DCIS and stromal cells from the same slide. CNV analysis of the two samples showed minimal CNV in normal stromal cells in contrast to DCIS where multiple CNVs were observed. Conclusions: Newly developed pulse laser retrieval system is suitable for capturing single cells for genomic analysis of breast cancer. WGA, WES, and CNV analysis was successfully carried out using the captured single cells and showed no difference in profile compared to those performed with bulk tissue. This method may have the potential to replace LCM for certain applications such as single cell analyses. Citation Format: Lee H-B, Kim S, Lee K-M, Jung Y, Lee AC, Kim J, Bae S, Ryu HS, Yoo T-K, Moon H-G, Noh D-Y, Kwon S, Han W. Genomic analysis of single cells isolated by a pulse laser retrieval system. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-02-01.

Tissue-Based Metabolomics to Analyze the Breast Cancer Metabolome.

Mass spectrometry and nuclear magnetic resonance-based metabolomics have been developed into mature technologies that can be utilized to analyze hundreds of biological samples in a high-throughput manner. Over the past few years, both technologies were utilized to analyze large cohorts of fresh frozen breast cancer tissues. Metabolite biomarkers were shown to separate breast cancer tissues from normal breast tissues with high sensitivity and specificity. Furthermore, the metabolome differed between hormone receptor positive (HR+) and hormone receptor negative (HR-) breast cancer, but was unchanged in HER2+ tumors compared to HER2- tumors. New metabolism-related biomarkers were discovered including the 4-aminobutyrate aminotransferase ABAT, where low mRNA expression led to an accumulation of beta-alanine and shortened relapse-free survival. The glutamate-to-glutamine ratio (GGR) represents another new biomarker that was increased in 88 % of HR- tumors and 56 % of HR+ tumors compared to normal breast tissues. The GGR might help to stratify patients for the treatment with specific glutaminase inhibitors that were recently developed and are currently being tested in phase I clinical studies. Surprisingly, 2-hydroxyglutarate (2-HG), initially found to accumulate in isocitrate dehydrogenase (IDH) mutated gliomas and leukemias and described as an oncometabolite, was detected to be drastically increased in several breast carcinomas in the absence of IDH mutations. In summary, metabolomics analysis of breast cancer tissues is a reliable method and has produced many new biological insights that may impact breast cancer diagnostics and treatment over the coming years.

Abstract A26: The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation as potential biomarkers for tamoxifen resistance

Abstract Background. The crosstalk between estrogen receptor alpha (ERα) and growth factor receptors is an essential part of tamoxifen resistance and the vascular endothelial growth factor/ tyrosine kinase domain receptor (VEGF/KDR) signaling pathway is clinically significant in the mechanisms of this resistance. The aim of the present study was to analyze the association of distribution pattern of ERα expression, VEGFR2 expression level, ESR1 and KDR single nucleotide polymorphisms (SNPs) with tamoxifen response in hormone receptor-positive primary breast cancer. Material and methods. Formalin-fixed paraffin-embedded and fresh frozen breast cancer tissue of the primary tumor was obtained from 97 hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen at the Tomsk Cancer Research Institute. The distribution patterns of ERα expression and Ki-67 protein expression were determined by immunohistochemistry. The quantitative expression levels of VEGFR2 (CD309) and p-Akt473 was measured using a flow cytometry. TaqMan SNP assays were used for genotyping ESR1+30T&amp;gt;C (rs2077647), ESR1 2014G&amp;gt;A (rs2228480), KDR-604T&amp;gt;C (rs2071559) and KDR 1192G&amp;gt;A (rs2305948) polymorphisms. Progression-free survival (PFS) was used as end-point for survival analyses. Results. Patients who did not benefit from tamoxifen treatment (tamoxifen resistance group -TR) showed significantly higher heterogeneous ERα expression than tamoxifen sensitive patients (TS; 81.1% and 58.3% respectively, P = 0.021). We found that ESR12014A mutant allele carriers were more prevalent in TR patients than in TS group (26.3% vs. 8.0%, respectively, P = 0.009). Similarly, the KDR -604T allele of rs2071559 was statistically significant related with tamoxifen resistance (P = 0.034). However, the KDR 1192G allele of rs2305948 were significantly less common in the tamoxifen progressive group compared to the tamoxifen sensitive patients (38.0% vs. 90.0%, respectively, P = 0.035). In addition, the variant homozygote KDR 1192GG (vs. AA) of rs2305948 was associated with VEGFR2 expression (r = -0.524, p = 0.012). VEGFR2 expression levels also showed borderline significant correlation with p-Akt473 expression (r = 0.368, p = 0.070). The Kaplan-Meier survival analysis demonstrated a significant relationship of heterogeneous distribution of ERα expression with poor survival of tamoxifen treated patients (log-rank P = 0.009). Moreover, the PFS for the patients with the KDR -604TT genotypes of rs2071559 were worse than for the patients with the CC genotype (log-rank P = 0.123). Conclusion. The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation can serve as potential predictors for hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen and thus provide more information for optimizing patient's selection to tamoxifen adjuvant therapy. Citation Format: Nataliya Babyshkina, Sergey Vtorushin, Stanislav Patalyak, Tatyana Dronova, Elena Slonimskaya, Nadejda Cherdyntseva. The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation as potential biomarkers for tamoxifen resistance. [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 A26.

Abstract 2913: Glutamate-enriched breast cancer as new entity and target for metabolic drugs

Abstract In many tumor cells, glutamine in addition to glucose serves as important source of energy and molecular building blocks. The first step of glutamine utilization is conversion of glutamine to glutamate by one of the glutaminase isoenzymes. Recently, two new allosteric inhibitors of glutaminase activity, BPTES and 968, have been discovered and described in the literature. Metabolic drugs developed as chemical analogs of these agents are planned to enter clinical trials in 2014. To quantify the activation of glutamine metabolism and to define a preferential target population for the new metabolic drugs, we analyzed glutamine and glutamate in a large cohort of breast carcinomas. Glutamine and glutamate were quantified in a training set of 224 fresh-frozen breast cancer tissues and an independent validation set of 143 fresh-frozen breast cancer tissues using gas chromatography combined with time-of-flight mass spectrometry (GC-TOFMS). Glutamate was strongly increased in cancer tissues compared to normal tissues in the training set (fold change=8.2, p=7.3E-24), while glutamine did not change significantly. The strong increase of glutamate in cancer tissues could be validated in the validation set (fold change=6.8, p=4.0E-28). Comparing ER- breast cancer with ER+ breast cancer, glutamate was increased with fold change 1.4 (p=4.7E-05), while glutamine was decreased with fold change -1.7 (p=0.0026) in the training set. Both changes could be validated in the validation set (glutamate: fold change=1.7, p=4.1E-07, glutamine: fold change=-2.6, p=0.00055). In the following, we used the glutamate/glutamine ratio (GGR) as biomarker for the activation of glutamine catabolism. In the training set, GGR was significantly higher in ER+ tumors compared to normal tissues (fold change=5.4, p=4.0E-21) and significantly higher in ER- tumors compared to ER+ tumors (fold change=2.4, p=8.5E-05). This pattern of deregulation could be validated in the validation set (fold change=7.3, p=4.0E-14 and fold change=4.4, p=1.9E-05). Higher GGR borderline significantly shortened overall survival in the training set (HR=1.31, p=0.09) and in the validation set (HR=1.32, p=0.13). In the combined set, correlation of overall survival and GGR was significant (HR=1.30, p=0.027). Using the training set data, we determined a cutoff value GGR=2 by requiring all normal tissues to be below the cutoff. In the validation set, only 2 of the 55 normal tissues (3.6%) were above the cutoff. In the combined set, the percentage of tissues above the cutoff increased from 2.2% in normal tissues via 57.1% and 55.5% in ER+/HER+ and ER+/HER2- tumors to 93.8% and 86.0% in ER-/HER+2 and ER-/HER2- tumors. In summary, we found an activated glutamine metabolism in about 90% of the ER- tumors and about 55% of the ER- tumors. These tumor populations showed an enrichment of glutamate relative to glutamine and represent a preferential target population for treatment with the newly developed glutaminase inhibitors. Citation Format: Jan Budczies, Berit M. Pfitzner, Klaus-Jürgen Winzer, Cornelia Radke, Manfred Dietel, Oliver Fiehn, Carsten Denkert. Glutamate-enriched breast cancer as new entity and target for metabolic drugs. [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 2913. doi:10.1158/1538-7445.AM2014-2913

OLC1 is overexpressed in breast cancer and its expression correlates with poor patient survival.

The overexpressed in lung cancer 1 (OLC1) has been demonstrated to be associated with numerous biological and pathological processes. However, the role of OLC1 in breast cancer has not been thoroughly elucidated. The purpose of this study was to assess OLC1 expression and to explore its contribution to the breast cancer. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted to detect OLC1 messenger RNA (mRNA) expression in 45 pairs of fresh-frozen breast cancer tissues and corresponding noncancerous tissues. Immunohistochemistry was performed to detect the expression of OLC1 in 145 breast cancer tissues. The relationship between the expression of OLC1 and clinicopathological characteristics and prognosis was statistically analyzed. We found that the expression levels of OLC1 mRNA and protein in breast cancer tissues were significantly higher than those in adjacent noncancerous tissues (P < 0.001). In addition, OLC1 expression was significantly correlated with tumor size (P = 0.034), grade (P = 0.015), stage (P < 0.001), and lymph node metastases (P = 0.028). Kaplan-Meier survival analysis showed that a high expression level of OLC1 resulted in a significantly poor prognosis of breast cancer patients. Further, Cox multivariate analysis indicated that OLC1 expression level was an independent prognostic factor for the overall survival rate of breast cancer patients. These findings provide evidence that a high expression level of OLC1 serves as a biomarker for poor prognosis for breast cancer. Thus, we speculate that OLC1 may be a potential target of antiangiogenic therapy for breast cancer.

MRNA expression of DOK1-6 in human breast cancer.

To examine the expression of downstream of tyrosine kinase (DOK)1-6 genes in normal and breast cancer tissue and correlated this with several clinico-pathological and prognostic factors. DOK1-6 mRNA extraction and reverse transcription were performed on fresh frozen breast cancer tissue samples (n = 112) and normal background breast tissue (n = 31). Tissues were collected between 1991 and 1996 at two centres and all patients underwent mastectomy and ipsilateral axillary node dissection. All tissues were randomly numbered and the details were only made known after all analyses were completed. Transcript levels of expression were determined using real-time polymerase chain reaction and analyzed against TNM stage, tumour grade and clinical outcome over a 10-year follow-up period. DOK-2 and DOK-6 expression decreased with increasing TNM stage. DOK-6 expression decreased with increasing Nottingham Prognostic Index (NPI) [NPI-1 vs NPI-3 (mean copy number 15.4 vs 0.22, 95%CI: 2.7-27.6, P = 0.018) and NPI-2 vs NPI-3 (mean copy number 7.6 vs 0.22, 95%CI: 0.1-14.6, P = 0.048)]. After a median follow up period of 10 years, higher levels of DOK-2 expression were found among patients who remained disease-free compared to those who developed local or distant recurrence (mean copy number 3.94 vs 0.0000096, 95%CI: 1.0-6.85, P = 0.0091), and distant recurrence (mean copy number 3.94 vs 0.0025, 95%CI: 1.0-6.84, P = 0.0092). Patients who remained disease-free had higher levels of DOK-6 expression compared to those who died from breast cancer. Decreasing expression levels of DOK-2 and DOK-6 with increased breast tumour progression supports the notion that DOK-2 and DOK-6 behave as tumour suppressors in human breast cancer.

Evidence for Tumour Suppressor Function of DOK7 in Human Breast Cancer

Introduction: Downstream of tyrosine kinase 7 (DOK-7) is a member of the DOK family, which has been associated with the development and progression of various humancancers. Previously, identification of CpG hypermethylation in DOK-7 promoter was identified in breast cancer. Method: DOK-7 mRNA extraction and reverse transcription were performed on fresh frozen breast cancer tissue samples and normal background breast tissue. Transcript levels of expression were analyzed against TNM stage, tumour grade and clinical outcome over a 10-year follow-up period. Results: Levels of DOK-7 expression decreased significantly with increasing TNM stage. Higher DOK-7 expression was correlated with longer disease free and overall survival times. Conclusion: To our knowledge, this is the first study to investigate DOK-7 expression in human breast cancer. We identify a potential DOK-7 tumour suppressor role. DOK-7 as a prognostic biomarker in human breast cancer should be included in future validation studies.

Correlation of microarray-based breast cancer molecular subtypes and clinical outcomes: implications for treatment optimization

BackgroundOptimizing treatment through microarray-based molecular subtyping is a promising method to address the problem of heterogeneity in breast cancer; however, current application is restricted to prediction of distant recurrence risk. This study investigated whether breast cancer molecular subtyping according to its global intrinsic biology could be used for treatment customization.MethodsGene expression profiling was conducted on fresh frozen breast cancer tissue collected from 327 patients in conjunction with thoroughly documented clinical data. A method of molecular subtyping based on 783 probe-sets was established and validated. Statistical analysis was performed to correlate molecular subtypes with survival outcome and adjuvant chemotherapy regimens. Heterogeneity of molecular subtypes within groups sharing the same distant recurrence risk predicted by genes of the Oncotype and MammaPrint predictors was studied.ResultsWe identified six molecular subtypes of breast cancer demonstrating distinctive molecular and clinical characteristics. These six subtypes showed similarities and significant differences from the Perou-Sørlie intrinsic types. Subtype I breast cancer was in concordance with chemosensitive basal-like intrinsic type. Adjuvant chemotherapy of lower intensity with CMF yielded survival outcome similar to those of CAF in this subtype. Subtype IV breast cancer was positive for ER with a full-range expression of HER2, responding poorly to CMF; however, this subtype showed excellent survival when treated with CAF. Reduced expression of a gene associated with methotrexate sensitivity in subtype IV was the likely reason for poor response to methotrexate. All subtype V breast cancer was positive for ER and had excellent long-term survival with hormonal therapy alone following surgery and/or radiation therapy. Adjuvant chemotherapy did not provide any survival benefit in early stages of subtype V patients. Subtype V was consistent with a unique subset of luminal A intrinsic type. When molecular subtypes were correlated with recurrence risk predicted by genes of Oncotype and MammaPrint predictors, a significant degree of heterogeneity within the same risk group was noted. This heterogeneity was distributed over several subtypes, suggesting that patients in the same risk groups require different treatment approaches.ConclusionsOur results indicate that the molecular subtypes established in this study can be utilized for customization of breast cancer treatment.

Higher expression levels of SOCS 1,3,4,7 are associated with earlier tumour stage and better clinical outcome in human breast cancer

BackgroundSuppressors of cytokine signaling (SOCS) are important negative feedback regulators of the JAK/STAT signaling pathway, and have been recently investigated for their role in the development of different cancers. In this study, we examined the expression of SOCS1-7 genes in normal and breast cancer tissue and correlated this with several clinico-pathological and prognostic factors.MethodsSOCS1-7 mRNA extraction and reverse transcription were performed on fresh frozen breast cancer tissue samples (n = 127) and normal background breast tissue (n = 31). Transcript levels of expression were determined using real-time PCR and analyzed against TNM stage, tumour grade and clinical outcome over a 10 year follow-up period.ResultsSOCS1,4,5,6 and 7 expression decreased with increased TNM stage (TNM1 vs. TNM3 p = 0.039, TNM1 vs. TNM4 p = 0.016, TNM2 vs. TNM4 p = 0.025, TNM1 vs. TNM3 p = 0.012, and TNM1 vs. TNM3 p = 0.044 respectively). SOCS2 and 3 expression decreased with increased Nottingham Prognostic Index (NPI) (NPI1 vs. NPI3 p = 0.033, and NPI2 vs. NPI3 p = 0.041 respectively). SOCS7 expression decreased with higher tumour grade (Grade 3 vs. Grade 2 p = 0.037). After a median follow up period of 10 years, we found higher levels of SOCS1,2 and 7 expression among those patients who remained disease-free compared to those who developed local recurrence (p = 0.0073, p = 0.021, and p = 0.039 respectively). Similarly, we found higher levels of SOCS 2,4, and 7 expression in those who remained disease-free compared to those who developed distant recurrence (p = 0.022, p = 0.024, and p = 0.033 respectively). Patients who remained disease-free had higher levels of SOCS1 and 2 expression compared to those who died from breast cancer (p = 0.02 and p = 0.033 respectively). The disease free survival (DFS) and overall survival (OS) curves showed that higher levels of SOCS1, 3 and 7 were significant predictors of higher DFS (p = 0.015, p = 0.024 and 0.03 respectively) and OS (p = 0.005, p = 0.013 and p = 0.035 respectively). Higher levels of SOCS 4 were significant in predicting better OS (p = 0.007) but not DFS. Immunohistochemical staining of representative samples showed a correlation between SOCS1, 3, 7 protein staining and the SOCS1, 3, 7 mRNA expression.ConclusionHigher mRNA expression levels of SOCS1, 3, 4 and 7 are significantly associated with earlier tumour stage and better clinical outcome in human breast cancer.

Genomic alterations identified by array comparative genomic hybridization as prognostic markers in breast cancer recurrence

11105 Background: We tried to establish models to predict systemic recurrence in breast cancer through the selection of the marker clones related with systemic recurrence by the analysis of array comparative genomic hybridization (array CGH). Methods: We performed array CGH with 4,044 human bacterial artificial chromosome (BAC) clones to assess copy number changes in 31 pairs of clinicopathologically well matched recurred/nonrecurred fresh-frozen breast cancer tissues. We selected those clones that hit the conditions of frequency criteria (more than 10%), consecutive clones (more than 2 clones), chi square test (p<0.05), multiple comparison test (False Discovery Rate (FDR), adjusted p<0.05), and Kaplan-Meier test (p<0.05). Results: 14 significant clones (11 clones in gain group and 3 clones in loss group) by disease free survival (DFS) analysis and 23 significant clones (17 clones in gain group and 6 clones in loss group) by FDR analysis were selected to be the candidate clones for predictive markers of systemic breast cancer recurrence. The most significant chromosomal alterations were found in the region of 5p15.33, 11q13.3, 15q26.3, 17q25.3, 18q23, 21q22.3 in gain, and 9p12, 11q24.1, 14q32.33 in loss. We presented the prediction models for systemic recurrence of breast cancer with 14 clones by DFS analysis and 23 clones by FDR analysis and the most effective cutoff values that can differentiate recurrence and nonrecurrence group were 9 and 16, respectively. Both methods are very effective in differentiation of recurrence and nonrecurrence group in ROC (Receiver operating characteristic) curve, DFS (disease free survival) curve, and OS (overall survival) curves analysis. Conclusion: New prediction models for systemic breast cancer recurrence by array CGH are very effective in differentiation of recurrence and nonrecurrence group. Candidate clone list by DFS analysis. Gain/loss Clone No FDR* DFS† OS‡ Cytoband Kb start Kb end Gain G1 0.006 0.0091 NS§ 18q23 75560433 75675514 G2 0.003 0.0101 NS 18q23 75297452 75415757 G3 0.012 0.0108 NS 5p15.33 473194 562560 G4 0.038 0.0141 NS 11q13.3 68979948 69057580 G5 0.006 0.0156 NS 21q22.3 45569815 45705137 G6 0.007 0.0159 NS 21q22.3 45471798 45579272 G7 0.015 0.0178 NS 18q23 75349325 75445847 G8 0.025 0.0256 NS 5p15.33 1238863 1353967 G9 0.010 0.0300 NS 15q26.3 100054894 100129431 G10 0.022 0.0325 NS 21q22.3 45997499 46084397 G11 0.038 0.0402 NS 17q25.3 78397724 78495803 G12 0.006 NS NS 21q22.3 46189033 46296404 G13 0.022 NS NS 21q22.3 46373142 46463667 G14 0.028 NS NS 21q22.3 45096044 45190446 G15 0.032 NS NS 7q36.3 157949176 158051524 G16 0.034 NS NS 21q22.3 45592803 45703201 G17 0.043 NS NS 21q22.3 45750504 45902278 Loss L1 0.011 0.020 NS 11q24.1 123006292 123091777 L2 0.024 0.022 0.019 14q32.33 105821330 105907464 L3 0.026 0.039 NS 9p12 43575387 43660307 L4 0.033 NS NS 8p23.1 7323700 7428919 L5 0.043 NS NS 8p23.3 318316 432795 L6 0.044 NS NS 10q11.22 46749417 46837491 * False discovery rate; † Disease free survival; ‡ Overall survival; § not significant Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Ministry of Health & Welfare, R.O.K

Influence of cytological stains on comparative genomic hybridization analysis for DNA extracted from cytological smears

In the present study, we investigated the influence of cytological stains in analyzing DNA extracted from cytological slides by comparative genomic hybridization (CGH). Multiple imprint cytological slides were prepared for fresh-frozen breast cancer tissue samples and the slides were stained by three staining methods for each sample. Under microscopic observation, cancer cells were selectively microdissected from the slides and forwarded to DNA extraction, whole genome amplification, and CGH analysis. CGH was successfully performed for all methylgreen-stained and May-Grunwald-Giemsa (MGG)-stained cytological smear slides, but for two Papanicolaou (PAP)-stained slides. The number of chromosomal imbalances detected were 5-10 in methylgreen-stained slides and 5-9 in MGG-stained slides. The chromosomal imbalances resemble each other between methylgreen-stained and MGG-stained slides. The present study indicates that the MGG stain is preferred to the PAP stain for the purpose of cytogenetical analysis by CGH for DNA extracted from cytological smear slides.

RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces

BackgroundAssessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1) Manual method = subjective evaluation of the electropherogram, 2) Ratio Method = the ratio between the 28S and 18S peaks, and 3) RNA integrity number (RIN) method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping.ResultsComparing the methods pair-wise, Manual vs. Ratio showed concordance (good vs. degraded RNA) in 20/24, Manual vs. RIN in 23/24, and Ratio vs. RIN in 21/24 samples. All three methods were concordant in 20/24 samples. The comparison between RNA quality and gene expression analysis showed that pieces from the same tumor and with good RNA quality clustered together in most cases, whereas those with poor quality often clustered apart. The number of samples clustering in an unexpected manner was lower for the Manual (n = 1) and RIN methods (n = 2) as compared to the Ratio method (n = 5).Assigning the data into two groups, RIN ≥ 6 or RIN < 6, all but one of the top ten differentially expressed genes showed decreased expression in the latter group; i.e. when the RNA became degraded. Ontological mapping using GoMiner (p ≤ 0.05; ≥ 3 genes changed) revealed deoxyribonuclease activity, collagen, regulation of cell adhesion, cytosolic ribosome, and NADH dehydrogenase activity, to be the five categories most affected by RNA quality.ConclusionThe results indicate that the Manual and RIN methods are superior to the Ratio method for evaluating RNA quality in fresh frozen breast cancer tissues. The objective measurement when using the RIN method is an advantage. Furthermore, the inclusion of samples with degraded RNA may profoundly affect gene expression levels.

Expression of estrogen and progesterone receptors in carcinomas of the female breast in Tanzania.

In Africa breast cancer has been reported to occur frequently in young females and to show an aggressive histological and clinical picture, suggesting that this malignancy might have a different biology from this disease in Western females. To investigate this, the present study assessed by immunohistochemistry the expression of estrogen receptors (ER) and progesterone receptors (PgR) in 60 fresh frozen breast cancer tissues from indigenous Tanzanian patients. This prospective study collected tissues from routine patients treated at the Muhimbili Medical Center, Dar es Salaam, Tanzania. These markers have not been previously investigated in indigenous sub-Saharan females with breast cancer. Patients in this study expressed lower frequencies of ER (33%) and PgR (18%) as compared to literature reports including those about African-Americans. Expression of these markers, however, correlated with the demographic, clinical and histological characteristics in a similar way as observed elsewhere. A compounding effect of younger patients' age, advanced disease or late stage at hospital presentation and race in this geographical region could be responsible for the poor expression of hormonal receptors in the majority of patients as observed in this study. A surprising finding was that the proportion of hormonal receptor positive tumors increased with disease duration. In view of the low frequency of expression of hormonal markers, only 26.7% of the patients would be expected to benefit from hormonal therapy based on their expression of the hormone receptors. There is great need to undertake an inter-African study that would evaluate the hormonal status of more African women with breast cancer in different geographical regions of sub-Saharan Africa and document the true picture of their hormonal status. The outcome of these results could be important for treatment strategies for the second most common cancer among African women.