bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential chemosensitivity to gemcitabine by cyclin E overexpression in human gastric cancers
Authors: Yeh K. , Shen Y. , Chiang Y. , Liu P. , Huang C. , Cheng A.
Published: March 2020

21020

Background: Exploration of molecular determinants for chemosensitivity is the key element of personalized cancer therapy. Cyclin E is a major G1-phase cyclin, together with the CDK2, which mediates phosphorylation and functional inactivation of Rb protein. Cyclin E overexpression has been demonstrated in a variety of cancers, including human gastric cancers (GC), while its biologic significance in drug therapy remains unclear. Previously, we have demonstrated that cyclin D1 overexpression plays an important role in differential chemosensitivity of human GC cells (Proc Am Assoc Cancer Res 2004; 45: abstract 4888). In this study, we examine the roles between cyclin E overexpression and chemosensitivity of human GC cells. Methods: Compared human gastric cancer cells (NCI-N87) with stably transfected cells (N87-CyE), which have 2-fold overexpression of cyclin E, the IC50 for gemcitabine (2'-2'-difluoro- deoxycytidine, dFdC) was more than 1-log lower in N87-CyE (17.0 ± 2.7 nM) than N87 cells (113.7 ± 6.5 nM) by MTT colorimetric cytotoxicity assay. In contrast, the N87-CyE cells are only slightly more chemosensitive to taxanes (paclitaxel and docetaxel), and confer largely identical chemosensitivity to 5-FU, cisplatin, and irinotecan (CPT-11). We applied RNA interference (RNAi) of cyclin E to N87 cells. The stably transfected N87-CyE/RNAi cells readily confer gemcitabine resistance with the IC50 for gemcitabine of 124.5 ± 1.6 nM. Results: Gemcitabine-induced apoptosis in N87, N87-CyE, or N87-CyE/RNAi cells was shown in either caspase-3 or PARP (poly ADP-ribose polymerase) cleavage assay by Western blotting, and Annexin-V-FITC apoptosis detection by flowcytometry. The threshold concentration of gemcitabine for caspase-3 and PARP cleavage was 10–25 nM for N87-CyE, and 100–200 nM for N87 or N87-CyE/RNAi, respectively. Conclusions: Our data demonstrate that preferential chemosensitivity to gemcitabine by cyclin E overexpression in gastric cancer cells. Gemcitabine-induced apoptosis is enhanced by cyclin E overexpression, while is reduced by RNAi of cyclin E. Further studies for potential clinical use of gemcitabine in personalized chemotherapy for cyclin E or cyclin D1-overexpressing GC are warranted (supported by the grants of NHRI-CN-CA9201S, Taiwan).

No significant financial relationships to disclose.
Relationship of angiogenesis biomarkers and clinical response to bevacizumab (Bev) in persistent or recurrent epithelial ovarian cancer (EOC) and primary peritoneal cancer (PPC) patients treated in a Phase II Gynecologic Oncology Group study
Authors: Han E. S. , Burger R. A. , Randall-Whitis L. , Parmakhtiar B. , Darcy K. , Sill M. , Connelly P. , Monk B. J. , Fruehauf J. P.
Published: March 2020

21021

Background: Angiogenesis promotes tumor growth and is associated with poor outcomes in ovarian cancer. Currently, there are no markers to predict or determine response to anti-angiogenesis therapies. Methods: Persistent or recurrent EOC/PPC patients (1–2 prior cytotoxic regimens, measurable disease, and GOG performance status = 2) were treated with Bev (15mg/kg IV q21days) until progression. Tissue biopsies, serum samples and dynamic contrast enhanced MRIs (DC-MRI) were obtained prior to initiation (PT01) and 4th cycle (PT02) of Bev therapy. Tumor vascular endothelial growth factor (VEGF), thrombospondin-1 (TSP-1), and mutant p53 were determined by immunohistochemistry (IHC) and scored using histoscore (HS) analysis ([1 + staining score (0 to 4)] x % positive). Microvessel density (MVD) was determined by counting CD31 IHC hot spots. Serum VEGF levels were obtained by ELISA. Variables were considered as continuous or discontinuous in this training set to establish appropriate cut points to be applied to an ongoing phase III trial (GOG218). Results: From 4/02 to 8/04, 62 eligible patients were enrolled. A 17.7% response rate with 40% of patients with no evidence of disease progression for at least 6 months was previously reported (ASCO 2005). 44 tissue samples from PT01 and 27 from PT02 were evaluable for IHC biomarker levels. Based on current analysis, VEGF HS average was 92 at low MVD counts, while at high MVD counts, VEGF HS average was higher at 159 (p=0.06). In addition, 77% of TSP-1 negative tissues were VEGF positive, while 52% of TSP-1 positive tissues were VEGF positive. Analysis of serum VEGF levels (from 56 PT01 and 39 PT02 specimens) and DCMRIs (49 PT01 and 42 PT02 MRIs) are ongoing. Conclusions: Higher tumorVEGF was associated with increased angiogenesis and lower TSP-1 tissue levels. Results from the biomarkers tested await correlation to the mature survival data which should be available by June 2007. We plan to determine whether an angiogenesis biomarker profile is associated with patient response to Bev therapy.

No significant financial relationships to disclose.
Epidermal growth factor receptor (EGFR) as a potential new target in the treatment of patients with adrenocortical carcinoma: Results of pre-clinical studies
Authors: Fassnacht M. , Hahner S. , Adam P. , Heinrich B. , Quinkler M. , Stroebel P. , Allolio B.
Published: March 2020

21025

Background: Adrenocortical carcinoma (ACC) is a rare malignancy with incompletely understood pathogenesis and poor prognosis. Overexpression of epidermal growth factor receptor (EGFR) has been demonstrated in several tumors and is partly associated with a more aggressive phenotype and a worse prognosis. In addition, targeting the EGFR tyrosine kinase represents a successful new therapeutic strategy, e.g. in non-small cell lung cancer. Therefore, we investigated the role of EGFR in ACC as a potential therapeutic target. Methods: EGFR expression was analyzed by immunohistochemistry in 115 ACCs and 5 normal adrenals using paraffin sections and tissue arrays (scoring of expression: 0–3). Utilizing the clinical data from the German ACC registry, Kaplan Meier survival analyses were performed. In 30 patients the tumor DNA was sequenced for mutations of the “hot spot” exons 19–21 of the EGFR gene. In addition, cells of the ACC cell line NCI-h295 were incubated with the EGFR antibody cetuximab (1–100 μg/ml) and cell proliferation was measured by MTT tests. Results: Immunohistochemistry revealed EGFR expression in 78% of ACCs. In 67/115 (58%) of the ACCs and 0/5 of the normal adrenals the expression level was judged as moderate-to-high (score 2 or 3). However, the expression level did not correlate with the clinical outcome in these patients. In addition, none of the sequenced tumor DNA samples showed a mutation in exons 19–21. Cetuximab exhibited a dose dependent antiproliferative effect in NCI-H295 cells (cell viability: 1μg/ml: 95±2%; 10μg/ml 90±3%*; 100 μg/ml 85±4%* vs untreated control cells: 100±3%; * = p<0.01). Conclusion: EGFR is overexpressed in the majority of ACC. Moreover, in vitro experiments demonstrated that inhibition of EGFR signalling lead to moderate growth inhibition in ACC cells. Therefore, in patients with ACC refractory to established cytotoxic therapies the experimental use of EGFR inhibitors (combined with cytotoxic therapy) seems to be justified.

No significant financial relationships to disclose.
Immune signatures hold prognostic import across solids tumors
Authors: Hsu S. D. , Anders C. K. , Acharya C. R. , Zhang Y. , Wang Y. , Foekens J. A. , Blackwell K. L. , Drake C. G. , Morse M. A. , Febbo P. G.
Published: March 2020

21041

Background: The host immune response can impact cancer growth, prognosis, and response to therapy. In colorectal cancer, the presence of cells involved with T-cell mediated adaptive immunity better predicts of survival than the current staging method. Immune signatures based on host response to cancer have the potential to predict prognosis and facilitate target specific therapy. Methods: We used the gene expression data associated with immune host response to colorectal cancer (Galon et al., Science 2006) to perform hierarchical clustering of solid tumors for which the clinical annotation were available. Specifically, prostate (n=79), breast (n=132), lung (n=37), and lymphoma (n=127) samples were clustered based upon expression of genes associated with host immune responses (Th1 mediated adaptive immunity (Th1), inflammation, and immune suppression (IS)). Kaplan-Meier survival analysis was then used to determine if major sample clusters had significantly different disease free survival. Results: Clusters with differential prognosis (disease free survival) were consistently seen across all tumors. Among adenocarcinomas, and similar to previously published colorectal data, the Th1 genes were consistently associated with better prognosis. Specifically, in breast cancer patients, increased expression of the Th1 genes was protective, but only in patients under 45 years of age (HR=0.42; p=0.05). In prostate cancer, patients with increased expression of the Th1 genes and inflammation genes had better prognosis when compared to those without the Th1 genes (HR=0.36; p=0.03) or inflammation genes (HR=0.37; p=0.03). Lung cancer patients expressing the Th1 genes and IS genes appear to have improved survival when compared to those without the IS genes (HR=0.33; p=0.08). In contrast, Th1 genes were associated with poorer prognosis in lymphoma patients. Patients with decreased Th1 genes and increased expression of inflammation genes had better prognosis than those expressing the Th1 genes (HR=0.43; p=0.013) or IS genes (HR= 0.35; p=0.002) only. Conclusions: Signatures of the host immune response to solid tumors hold prognostic significance. Future work will determine if immune signatures can be predictive of response to therapy and help guide management.

No significant financial relationships to disclose.
Oncolytic adenovirus expressing apoptotic genes targets radiation resistant (RR) esophageal cancer
Authors: Chang J. Y. , Komaki R. , Zhang X. , Wang L. , Fang B.
Published: March 2020

21043

Background: Only 25% of esophageal cancer patients achieve pathological complete response after standard chemoradiotherapy. Radiation dose escalation is associated with higher toxicity but no therapeutic improvement. In addition, esophageal cancer cells may develop radiation resistance (RR) after fractionated radiation exposure. Therefore, molecular targeting therapy for RR esophageal cancer is urgently needed. Methods: Six pairs of RR esophageal cancer cell lines were established by applying continuous 2 Gy fractionated irradiation. Ad/TRAIL-E1, an oncolytic adenoviral vector expressing both apoptotic TRAIL and viral E1A genes under the control of tumor specific human telomerase reverse transcriptase promoter, was constructed. Phosphate buffer solution and vectors expressing the TRAIL gene only, the GFP marker protein only, or the E1A gene only served as controls. Trans-gene expression, apoptosis activation, and the RR esophageal cancer cells targeted were evaluated in vitro and in vivo. A human esophageal RR cancer model was established and locally treated with Ad/TRAIL-E1 or controls. Results: After fractionated radiation exposure, esophageal cancer cell lines developed RR (up to 25-fold) that was associated with activation of the anti-apoptotic pathway. Ad/TRAIL-E1 activated an apoptotic cascade of caspases and selectively killed esophageal cancer cells but not normal cells. Ad/TRAIL-E1 preferentially targeted RR stem-like cancer cells with higher trans-gene expression and cell killing compared with parental cells. Overexpression (3 times) of Coxsackie's and adenoviral receptors in RR esophageal cancer cells compared with parental cells was noted. Ad/TRAIL-E1 therapy resulted in 40% tumor-free survival without the treatment- related toxicity found in human RR esophageal adenocarcinoma mouse models (p<0.05 as compared with controls). Conclusions: Esophageal cancer cells develop RR after fractionated radiation exposure. Ad/TRAIL-E1 preferentially targeted RR stem-like esophageal cancer cells, which resulted in a 40% cure rate.

No significant financial relationships to disclose.
The role of Notch3 signaling pathway in pancreatic cancer
Authors: Dang T. , Vo k. , Washington K. , Berlin J.
Published: March 2020

21049

Background: Given the extremely poor prognosis of patients diagnosed with pancreatic cancer, identifying new targets for therapeutic intervention is crucial in improving clinical outcome. Existing data support a role for Notch3 pathway in human cancer, pancreas cancer in specific and in normal pancreas development. Method: We developed a pancreatic tissue array. An antibody targeting the extracellular domain of Notch3 was used to evaluate expression levels, grading from 0 to 4+ to determine the prevalence of Notch3 in pancreatic cancer. Notch3 expression was used to correlate with clinical data (IRB approved). Using small interfering RNA (siRNA) we tested the downstream effects of Notch3 in pancreatic cancer cell line BxP3 in vitro. Furthermore, to study pharmacologic inhibition of Notch3 processing and activation we exposed Panc-1, HPAF II and BxP3 pancreas cancer cell lines to γ-secretase inhibitors (previously demonstrated to block upstream of Notch3 thereby inhibiting Notch3). Results: Strong staining (+2 or greater) for Notch3 was seen in 50 of 72 (69%) resected specimens. No correlation with survival or tumor grade was seen. Using siRNA, we demonstrated that inhibiting Notch3 downregulated the antiapoptotic protein Bcl-xL but not Bcl-2. When γ-secretase inhibitors (GSI and L-685,458) were used, the previously mentioned pancreatic cell lines demonstrated reduction in proliferation rates. Conclusion: Notch3 overexpression is common in pancreas cancer. While Notch 3 expression does not clearly correlate with survival, our in vitro data suggest that Notch3 affects apoptotic pathways and may represent a potential target for intervention in patients with pancreatic cancer.

No significant financial relationships to disclose.
Relationship between polymorphisms in cordon 118 and C8092A in ERCC1 and clinical outcome in optimally-resected, stage III epithelial ovarian cancer treated with intraperitoneal or intravenous cisplatin and paclitaxel: A gynecologic oncology
Authors: Krivak T. C. , Darcy K. M. , Tian C. , Baysal B. E. , Armstrong D. K. , Gallion H. H. , Deloia J. A.
Published: March 2020

21050

Background: The association between polymorphisms in the excision nuclease ERCC1 and progression-free survival (PFS) or overall survival (OS) was examined in women with epithelial ovarian cancer (EOC) treated with cisplatin and paclitaxel. Methods: Women who were evaluable for the phase III trial, GOG-172, with sufficient leukocyte DNA for testing were eligible for this study. Participants were randomized to intraperitoneal (IP) or intravenous (IV) cisplatin and paclitaxel. Single nucleotide polymorphism (SNP) analysis was carried out by direct pyrosequencing. Results: Among the 233 eligible cases, the genotype distribution at codon 118 was 17% with C/C, 43% with C/T and 40% with T/T and at C8092A was 56% with C/C, 37% with C/A and 7% with A/A. Adjusted Cox regression analysis revealed that the codon 118 polymorphism was not significantly associated with disease progression or death, but women with the C/A or A/A genotype compared with the C/C genotype at C8092A had an increased risk of disease progression (hazard ratio [HR]=1.48, 95% confidence interval [CI]=1.09–2.00, p=0.011) and death (HR=1.46, 95% CI=1.04–2.04, p=0.029). Subset analysis stratified by treatment suggested that the C8092A polymorphism was significantly associated with increased risk of PFS and OS in the IP but not the IV arm. Conclusions: Although the ERCC1 codon 118 polymorphism does not appear to be associated with clinical outcome, the C8092A polymorphism in ERCC1 was an independent predictor of PFS and OS in optimally-resected EOC treated with cisplatin- paclitaxel chemotherapy. The preferential clinical utility of the C8092A polymorphism in the IP but not the IV arm requires validation.

No significant financial relationships to disclose.
Confirmation of the distinct molecular phenotype of inflammatory breast cancer compared to non-inflammatory breast cancer using Affymetrix-based genome-wide gene expression analysis
Authors: Van Laere S. J. , Van der Auwera I. , Van den Eynden G. G. , Trinh X. , Van Hummelen P. , van Dam P. , Van Marck E. A. , Vermeulen P. B. , Dirix L. Y.
Published: March 2020

21055

Background: We have shown with cDNA microarrays that inflammatory breast cancer (IBC) and non-IBC are distinct biological entities. The purpose of this study was to confirm our previous results using Affymetrix chips. Methods: RNA was extracted from 19 IBC samples and 42 non-stage matched non-IBC samples. RNA was hybridized onto Affymetrix HG U133 Plus 2.0 chips. Gene expression data were normalized using GCRMA and genes with a gene expression of at least 250 in 50% of the cases were filtered in. Hierarchical clustering and principle component analysis was executed. Identification of the different cell-of-origin subtypes in our expression data set was done using the intrinsic gene list. A NFkB signature, a MAPK signature and our own IBC signature were tested by clustering analysis. Results: Clustering using 11341 genes resulted in the identification of two clusters: one containing 14/19 IBC samples and a second containing 32/42 non-IBC (Pearson χ2; p<0.0001). Principle component analysis separated IBC from non-IBC samples along the first principle component. Interestingly, IBC samples more closely resemble T1 - T2 tumours than T3 - T4 tumours. Application of the intrinsic gene set to our IBC/non-IBC data set resulted in the classification of 14/19 IBC samples as basal-like or ErbB2-overexpressing tumours compared to only 4/42 non-IBC tumours (Pearson χ2; p<0.0001). Our own IBC signature was confronted with the new data set and performed well in separating IBC specimens form non-IBC specimens. Clustering identified three clusters from which one cluster contained 18 samples, including 12 IBC specimens (p<0.0001). Using the NFkB and MAPK signatures, similar results were obtained. Conclusions: These results confirm our findings that IBC is a distinct biologic phenotype, characterized by activation of NFkB, possibly through activation of MAPK's. IBC tumours more often demonstrate characteristics from basal-like and ErbB2-overexpressing breast tumours. The fact that IBC tumours are rapidly developing tumours instead of longstanding tumourigenic processes might explain the close resemblance of the IBC gene expression profile to the gene expression profile of T1 and T2 tumours.

No significant financial relationships to disclose.
Expression of the WAVE (WASP Verprolin-homologous) molecules in human breast cancer
Authors: Fernando H. , Chhabra A. , Davies S. , Watkins G. , Kynaston H. , Mansel R. E. , Jiang W. G. ,   Metastasis and Angiogensis Research Group
Published: March 2020

21061

Background: WAVEs (WASP Verprolin-homologous) belongs to the CCN family which comprises of nine members, the cysteine-rich 61 (Cyr61/CCN1), connective tissue growth factor (CTGF/CCN2) and nephroblastoma over-expressed (Nov/CCN3), Wnt-induced secreted proteins(WISPs)1, 2 and 3 (CCN4–6) as well as the WAVE-1,2 and 3 (also known as CCN7–9 respectively). Some members of the CCN family, such as Cyr61 and CTGF are known to stimulate mitosis, adhesion, apoptosis, extracellular matrix production, cell migration and growth arrest in cancer cells including breast cancer cells. However, there is no knowledge of the expression pattern and the function of WAVEs in human breast cancer. Here, we report the expression of WAVE-1,-2 and -3 transcripts and proteins in relation to clinical and pathological characteristics in human breast cancer. Methods: The expression of the three WAVE molecules at the mRNA and protein levels in a cohort of 122 human breast cancers and 32 normal breast tissues were analysed and correlated with the patients’ clinical outcome. The respective transcripts were quantitative determined using real time RT-PCR and distribution of the proteins were investigated by immunohistochemical methods. Results: All three WAVE proteins were detected in mammary epithelial cells and are of cytoplasmic nature. Breast cancer cells from the tissues also displayed positive staining of the WAVE proteins, with WAVE-2 staining appears weaker compared with normal epithelial cells. WAVE-1 transcripts were expressed in significantly high levels (p=0.03) in high grade breast cancers. Low levels of WAVE-2 were associated with higher TNM staging (p=0.038 in TNM3 and p=0.017 in TNM4) and were also found in patients with poor prognosis. Interesting, a marginal reduction of WAVE-3 transcripts was seen in patients who developed systemic metastasis. No other significant associations were found between the WAVE1 and WAVE3 transcripts and the breast cancer cells. Conclusions: WAVEs are widely expressed in human mammary tissues and have a differential expression in human breast cancer with WAVE2 appearing to be associated with the aggressiveness of breast tumors.

No significant financial relationships to disclose.
An association between thyroid disease and breast cancer
Authors: Karlin N. J. , Chopra I. , Mirocha J. , Feldman N.
Published: March 2020

21063

Background: Anecdotal studies and isolated case reports have observed an association of thyroid disease and breast cancer. This purported association remains controversial. We describe a retrospective case-control study on the prevalence of breast cancer in patients with abnormal thyroid function studies. Age-matched subjects served as controls. Methods: We reviewed 6211 cases of abnormal TSH values between 1/04 and 12/05 and determined the number of cases with breast cancer. We compared those data to 3,151 control subjects with normal TSH seen during the same period. We excluded patients with thyroid cancer from the total number of malignancies in both groups because abnormal TSH would be expected to result from its treatment. We also reviewed the ER, PR, her 2 neu status and free thyroxine of patients with breast cancers. Results: Breast cancer rate of 36% of all cancers (37/102) in the abnormal TSH group was significantly higher than that of 18% (14/77) in the normal TSH group. (p = 0.012). Breast cancers in the study group with abnormal thyroid function were more frequently hormone receptor positive (74% vs. 67%) and her 2 neu negative (67% vs. 50%). Conclusions: The data suggest that there is an increased rate of breast cancer in patients with abnormal thyroid function. The trend of hormone receptor positive disease in the abnormal TSH group suggests that the IGF receptor may play an important role in the relationship between thyroid disease and breast cancer. The IGF receptor may be a potential target for therapeutic drug development for breast cancer prevention and management. Further studies are warranted.

No significant financial relationships to disclose.
CCR5 gene polymorphism in patients with cutaneous melanoma: Impact on survival following immunotherapy
Authors: Becker J. C. , Ugurel S. , Zapatka M. , Kaufmann H. L. , Schrama D.
Published: March 2020

21071

Background: Chemokines are supposed to influence both tumor progression and anti-tumoral immune responses. In this regard, a 32-bp-deletion polymorphism in the chemokine receptor 5 gene (CCR5Δ32) resulting in a non-functional receptor protein allows to study the potential impact of the CCR5 system on the clinical course and treatment outcome of malignant diseases. Methods: CCR5 genotyping was performed by PCR on DNA extracted from serum samples of 782 cutaneous melanoma patients with known disease history and long-term clinical follow-up. Genotypes were correlated with patient survival in consideration of different therapeutic modalities. Results: Out of 782 patients, 90 (11.5%) were heterozygous and 12 (1.5%) were homozygous for CCR5Δ32. Disease-specific survival starting from primary diagnosis was not influenced by CCR5 status. Equally, no impact of the CCR5 status could be detected on treatment outcomes of stage III patients. In 139 stage IV patients treated with immunotherapeutics, however, CCR5Δ32 was associated with a diminished survival compared to patients not carrying the Δ32 allele (p=0.029). Multivariate analysis revealed the CCR5 genotype as an independent factor impacting disease-specific survival in this patient group (p=0.0084), followed by gender (p=0.017), and type of primary (p=0.029). Vice versa, stage IV patients without immunotherapy revealed a trend towards a favorable survival when harboring CCR5?32 (p=0.12). Conclusions: The disadvantage of CCR5Δ32-bearing stage IV melanoma patients with respect to outcome of immunotherapy indicates a strong impact of the CCR5 mediator system on anti-tumoral immune responses, and should be taken into account when choosing therapeutic modalities for these patients.

No significant financial relationships to disclose.
Use of nuclear androgen receptor status to predict early biochemical recurrence of prostate cancer patients
Authors: Diallo J. , Aldejmah A. , Alam Fahmy M. , Koumakpayi I. , Mes-Masson A. , Saad F.
Published: March 2020

21073

Background: Prostate cancer (PCa) is a leading cause of cancer death in North American men. The androgen receptor (AR) has an established role in the progression of this disease; however, it is unclear at what stage it intervenes. It is also uncertain whether the AR can be a useful prognostic marker for PCa. In this study, we assessed AR expression and sub-cellular localization in normal prostate as well as in androgen sensitive and insensitive PCa (AIPCa) tissues, and evaluated the ability of the AR to predict biochemical recurrence (BCR). Methods: We used tissue micro-arrays containing prostate tissue cores obtained from cancer-free patients (n=43), AIPCa patients (n=36), and patients with hormone-sensitive cancers (n=64) from which were collected both cancerous and normal adjacent tissue. Using immmunohistochemistry, we stained the tissue micro-arrays with a monoclonal antibody recognizing the AR. Two observers assessed the frequency and intensity of both cytoplasmic and nuclear AR staining. AR cytoplasmic (Ci) and nuclear (Ni) indices were calculated by multiplying nuclear staining frequency and nuclear staining intensity. Kaplan Meier, and Cox multivariate analyses were done using SPSS. Results: We found that AR Ci increased significantly in AIPCa although a modest but significant increase in PCa Ci was observed compared to normal tissues. In contrast, AR Ni was significantly lower in cancer-free patients as opposed to that seen in normal tissue adjacent to cancer. Similarly, cancerous tissue exhibited higher AR Ni than its adjacent normal tissue (p<0.05, Kruskal-Wallis). Kaplan Meier analyses revealed that low AR Ni was predictive of an early onset of BCR (before 3-years) in the sub-cohort of hormone-sensitive patients (LR=6.51, p=0.011). Futhermore, low AR Ni remained an independent predictor of early BCR in a Cox multivariate model controlling for age, pre-operative PSA, lymph node invasion, Gleason score and surgical margin status (HR=2.28, 95% CI=1.04 - 5, p<0.05). Conclusions: We conclude that increased nuclear AR activity could be a pre-malignant step in PCa progression whereas its role within cancer cells may be more complex, as low AR nuclear activity was associated with early onset of BCR.

No significant financial relationships to disclose.
Insulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 is active in breast cancer cells and enhances growth inhibition by herceptin through an increase in cell cycle arrest
Authors: Esparís-Ogando A. , Rodríguez-Barrueco R. , Borges J. , Ferreira L. , Pandiella A. , Ocana A.
Published: March 2020

21077

Background: Targeting ErbB2 with the monoclonal antibody trastuzumab (Herceptin) has shown to be active in breast cancer. However, a proportion of patients do not benefit from this treatment due to primary resistance. Mechanisms proposed for this resistance include activation of other receptors involved in proliferation as is the case of the insulin-like growth factor receptor (IGF-1R).Different strategies have been proposed for the inhibition of IGF-1R such as antibodies or small tyrosine-kinase inhibitors. In this study we analyzed the mechanism of action and the antiproliferative activity of an specific IGF-1R tyrosine-kinase inhibitor termed NVP-AEW541 alone and in combination with Herceptin. Methods: MCF7, SKBR3, BT474, and MDA-MB-231 breast cancer cells were used for this study. Proliferation and apoptosis were analyzed by MTT uptake assays or Annexin-V-FITC, respectively. The levels of different signalling proteins were analyzed by Western blotting. Microarray studies were performed using the HU-133A oligonucleotide arrays (Affymetrix, Santa Clara, CA). Real time quantitative PCR was used to confirm the differentially regulated genes. Results: The IGF-1R was expressed in all the cell lines studied. Exogenous addition of IGF-1 increased the tyrosine phosphorylation of IGF-1R, and this was prevented by preincubation with NVP- AEW541. The latter also decreased MTT uptake, and provoked a slight increase in apoptosis of BT474 cells. Combination of NVP-AEW541 and Herceptin had a synergistic effect on the inhibition of BT474 proliferation. Cell cycle analyses indicated that the combined addition of NVP- AEW541 and Herceptin increased the number of cells in the G0/G1 phases. These data were complemented with an increase in p27 levels upon treatment with both compounds. Microarray analyses identified several genes whose expression was modified by each agent alone and by the combination. Conclusions: Our results indicate that the combined targeting of two receptor tyrosine kinases known to play important roles in tumor cell proliferation may be more efficient than individual targeting of these receptors.

No significant financial relationships to disclose.
Zoledronic acid induces anti-cancer effects on prostate cancer cells through the modulation of the angiogenesis associated CYR61 and the androgen-differentiation associated NDRG-1 genes
Authors: Tonini G. , Vincenzi B. , Marra M. , Baldi A. , Addeo S. , Loeffler M. , Rosolowski M. , Budillon A. , Caraglia M. , Santini D.
Published: March 2020

21079

Background: Aminobisphosphonates (ABPs) has a definite direct anti-tumour activity but a limited activity in vivo. Their molecular targets are still not completely defined. Therefore, we have studied the effects of zoledronic acid (ZOL) addition to prostate cancer PC3 cells on gene expression profile. Methods: We have treated PC3 cells with 100 μM ZOL for 24 hours, extracted mRNAs and probed on Affimetrix HG-U133. Thereafter, we have identified down modulated and upregulated genes and checked for modulation of mRNA with RT PCR and of the relative encoded proteins with western blotting. Results: We have found 6 down modulated and 32 upregulated genes. We have focused our attention on NDRG1 associated to the androgen-differentiation and on Cysteine rich 61 (CYR61) involved in the regulation of proliferation and angiogenesis. NDRG1 mRNA was up-regulated and CYR61 mRNA was downregulated by ZOL in a dose-dependent manner. Similar effects were observed at protein product levels with an about 2-fold change recorded already in cells treated with 50 μM ZOL. Interestingly, also Gefitinib, Sorafenib and Tipifarnib used at their IC:50s could induce changes in both NDRG1 and CYR61 expression, but 50 μM ZOL was about 2-fold more potent. On the other hand, cytotoxic agents such as docetaxel did not have any effect. The addition of farnesol (FOH) or geranylgeraniol (GGOH) to ZOL-treated cells was able to counteract the effect of ZOL on CYR61 expression partially or completely, respectively. On the other hand, both FOH and GGOH had poor effect on the regulation of NDRG1 expression induced by ZOL. Conclusions: ZOL induces a strong regulation of the expression of NDRG1 and CYR61 at both mRNA and protein levels that appears to be dose-dependent and specific. CYR61 modulation seems to be more dependent from the inhibition of geranylgeranylation processes while NDRG1 changes could be at least in part independent from the inhibition of isoprenylation induced by ZOL. The study of the biological relevance of these effects on the anti-cancer effects of ZOL is ongoing with small interference RNA approaches.

No significant financial relationships to disclose.
Persistence of circulating tumour cells after treatment predicts clinical outcome in breast cancer patients
Authors: Serrano M. , Sánchez-Rovira P. , Campos M. , Warleta F. , Ruiz-Mora J. , Algarra I. , Delgado-Rodríguez M. , Fernández M. , de la Torre C. , Peiró A. , Gaforio J. J.
Published: March 2020

21080

Background: The hematogenous distant metastasis is the leading cause of cancer death. This process involves the passage through the blood and lymphatic circulation of circulating tumor cells (CTC) with metastatic properties. Thus, the early detection of such cells has important implication for cancer prognosis, monitoring of treatment and to predict clinical outcome. We present the results that showed that CTC are prognostic factor after chemotherapy independently of treatment. Methods: A total of 59 patients with breast cancer were enrolled in this study between April 2000 and December 2002. The median follow-up was 50 months. All patients received chemotherapy as first line treatment. Results of this work included CTC detection one month after of the end of chemotherapy. After informed consent, 10ml of heparinized peripheral blood was collected from patients. For enrichment of CTC we use the Carcinoma Cell Enrichment and Detection kit using MACS technology (Miltenyi Biotec). After enrichment of epithelial tumor cells immunomagnetic labeled with a multi- cytokeratin-specific antibody, the positive cells were detected by immunocytochemical staining with alkaline phosphatase substrate. Results: Analysis of CTC after chemotherapy: Circulating tumor cells were detected in 32 patients (54.23%). A mean number of cells were detected 3.7 (SD 13.9; range 1–105).The number of CTC was correlationed with progression free of disease (PFS) and overall survival (OS). In the univariate Cox proportional hazard regression analysis number cells were significantly associated with OS (p = 0.020) and PFS (p = 0.008). In the multivariate Cox proportional hazard regression analysis, number cells admit borderline statically significance with PFS (p = 0.052) and OS (p = 0.071). Conclusions: Our results suggest that the persistence of CTC after the treatment predicts clinical outcome and therefore the detection of CTC in breast cancer patients might allow monitoring of chemotherapy response.

No significant financial relationships to disclose.
Multiplexed genotyping using a novel digitally inscribed bead-based system
Authors: Lipkin S. M. , Yeakley J. , Chao E. , Velasquez J. , Lopez M. , Rhee J. , McDaniel T. , Lewis I. , Chen H. ,   Illumina Genotyping Study Group
Published: March 2020

21089

Background: Genotyping of clinical samples has been limited to low levels of multiplexing, ranging from one to a few dozen single nucleotide polymorphisms (SNPs) per sample. By increasing multiplexing levels, a clinical lab can increase information content per sample, decreasing costs and sample material requirements. Methods: We have adapted the GoldenGate® Assay for simultaneously genotyping 96 to 1,536 SNPs to the BeadXpress™ System, a new high-throughput platform that utilizes digitally inscribed VeraCode™ beads in a compact fluidic instrument. Genotyping on this platform ranges from 96 to 384 multiplexing, using the same GoldenGate Assay that has proven highly robust for millions of genotypes. In preliminary tests, we have observed greater than 99% call rates, and greater than 99.5% rates for reproducibility and heritability. In a test of 96 SNP genotypes chosen for a study of colorectal cancer, a point mutation in the MSH2 gene, previously implicated in predisposition to several cancers, was correctly genotyped when compared to qPCR analysis of the same samples. Conclusion: Together with genotyping data from reference samples, the GoldenGate Assay on the BeadXpress System has yielded highly reproducible and accurate genotypes, suggesting that this approach will prove useful for rapid refinement of SNPs for development of clinical genotyping tests.

No significant financial relationships to disclose.
Stem cell markers in gastrointestinal cancer
Authors: Valladares-Ayerbes M. , Díaz Prado S. , Medina V. , Iglesias P. , Rodríguez B. , Haz M. , Reboredo M. , Quindós M. , Alonso-Jaudenes G. , Antón Aparicio L.
Published: March 2020

21095

Background: Cancer cells with stem cells (CSC) properties have been identified in different tumors. It is suggested that CSC are responsible for the continuous growth of tumors, metastasis and drug-resistance. Markers for stem cells have been described. Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. ABCG2 transporter (MDR1) gene expression has been described as surrogate for the side-population phenotype. PTTG1 has also been recently identified as a component of the molecular signature of human (hu) ES cell-lines. Methods: Using Digital Northern we have demonstrated a significant tag counts for PTTG1 and reticulocalbin 2 (RCN2) in 11 huES cell-lines of the CGAP. The objective of our work has been to assess gene expression of these SC markers in a panel of new gastrointestinal cancer (GC) cells lines (CL) developed in our laboratory. Quantitative assessment was obtained by real-time PCR relative to normal bone marrow (BM), colonic mucosa and established cell-lines. GCCLs have been developed from ascitic fluid obtained of pancreatic carcinoma (MBQ-OJC1) and colon cancer (JJPF-OJC4, LCM-OJC5 and JAC-OJC6). GCCLs had been fully characterized by cytomorphology, epithelial and tumor markers (keratins, EGFR, EpCAM, p53), karyotype and tumor spheroids cultures. Results: Expression for ABCG2, Nanog, Oct4, PTTG1 and RCN2 were clearly detected in all the GCCL. Relative levels for each mRNA shown wide variety. For example, ABCG2 mRNA was highly expressed (2–26 fold) in colon cancer CL relative to BM. RCN2 was overexpressed (more than 2 x 102 fold) in 3 GCCL. Conclusions: Our results show that expressions of different “stemness” genes are maintained in cultured cancer cells. These data suggest that CSC are present in metastatic sites and can be maintained in continuous culture. We hypothesized that PTTG1 and RCN2 could be tested as a new cancer stem cells markers.

No significant financial relationships to disclose.
Prospective study of a 10-gene molecular assay to predict tissue of origin in patients with carcinoma of unknown primary (CUP)
Authors: Varadhachary G. R. , Talantov D. , Jatkoe T. , Rashid A. , Lenzi R. , Chadha R. , Baden J. , Wang Y. , Abbruzzese J. L. , Raber M. N.
Published: March 2020

21096

Background: Carcinoma of unknown primary (CUP) where metastatic disease presents without an identifiable primary represents ∼ 3–5% of all cancers. Identifying the origin of the primary tumor in CUP pts can facilitate rational choice of therapeutic regimens. Veridex developed an optimized set of 10 gene markers for a quantitative reverse transcriptase polymerase chain reaction (qRTPCR) assay, and demonstrated high accuracy in predicting the tissue of origin with formalin-fixed, paraffin-embedded (FFPE) metastatic carcinoma samples (J Mol Diagn 2006, 8: 320–9). In this study, the 10-gene assay was prospectively evaluated in CUP pts. Methods: We collected FFPE biopsy tissue specimens from consenting CUP pts at MD Anderson. Eligibile pts met our definition for CUP with adenocarcinoma or poorly differentiated carcinoma. Samples were obtained prior to treatment. 51 pts have been enrolled so far and 11 were ineligible [insufficient samples].Of the 40 pts, qRTPCR assay has been performed on 33 pts. Data on 27/33 is available. A statistical model was used to determine the probability that the metastatic carcinoma tissue assayed originated from 1 of the following 7 categories: lung, pancreas, colon, breast, prostate, ovarian, and other. Subsequently, prediction of the primary by qRTPCR was independently compared with metastatic pattern spread, tumor pathological features, and results of clinical and pathology diagnostic workups. Results: Assay results on 27 prospectively collected CUP patient biopsy specimens are available. In total, CUP tissue of origin prediction by the assay correlated with clinical and pathological assessment in 21 out of 27 evaluated pts (78 %). The most common cancer type predicted by the assay was colon cancer, which correlated with predominantly intra-abdominal metastatic spread in this pt cohort. Conclusions: This prospective study demonstrated the feasibility of conducting gene analysis to predict metastatic carcinoma tissue of origin in FFPE tissue specimens derived from CUP patients. Overall distribution of various primary cancer types as predicted by the assay was consistent with the historical distribution reported for CUP. Assay prediction was concordant with clinical and pathological assessment in 78 % CUP pts.

No significant financial relationships to disclose.
Tumor necrosis factor-α is a potent endogenous mutagen that promotes cellular transformation
Authors: Yan B. , Wang H. , Rabbani Z. N. , Dewhirst M. W. , Li C.
Published: March 2020

21104

Background: It is estimated that chronic inflammation is an important underlying condition for sporadic human tumor development, accounting for approximately 20% of human cancers. TNFa is an inflammation cytokine playing an important role in inflammation- associated cancers. However, the underlying mechanism of how TNFa leads to cancer is not clear. Before we initiated this study, TNFa is not known to have a direct effect on DNA. Methods: We studied genetic mutations, cellular transformation in cells and mice treated with TNFa. DNA damages and genetic aberrations caused by TNFa treatment were measured by gene amplification assay, micronucleus assay, cytogenetic analysis for chromosomal aberrations and immunostaining of 8-OHdG (8-hydroxydeoxyguanine; marker for DNA damage and oxidative stress). Cellular transformation was studied by standard transformation assay using mouse embryo fibroblast cells. Results: In this study, we found that TNFa can cause DNA damages through induction of reactive oxygen species (ROS). Our findings include: (1) The mutagenic effect of TNFa is comparable to that of ionizing radiation. TNFa treatment in cultured cells resulted in increased gene amplification, micronuclei formation and chromosomal instability. (2) Antioxidants significantly reduced TNFa-induced genetic mutations. (3) TNFa also induced oxidative stress and nucleotide damages in mouse tissues in vivo. (4) Moreover, TNFa treatment alone led to increased malignant transformation of mouse embryo fibroblasts, which could be partially suppressed by antioxidants. (5) The mutagenic effect of TNFa is independent that of NF?B which was shown to be a mediator in inflammation-associated cancers. Conclusions: As TNFa is known to be involved in chronic inflammatory diseases such as chronic hepatitis, ulcerative colitis and chronic skin ulcers and these diseases predispose the patients to cancer development, our results suggest a novel pathway through which TNFa promotes cancer development through induction of gene mutations, in addition to the previously reported mechanisms in which NF-?B activation was implicated.

No significant financial relationships to disclose.
Gene profiling of tumor tissue in the diagnosis of patients with carcinoma of unknown primary site (CUP): Evaluation of the Veridex 10-gene molecular assay
Authors: Hainsworth J. D. , Talantov D. , Jatkoe T. , Meng C. , Baden J. , Spigel D. R. , Mazumder A. , Wang Y. , Greco F. A.
Published: March 2020

21109

Background: Standard treatment for most patients with CUP involves empiric chemotherapy. Since specific treatment now exists for most types of advanced carcinoma, precise identification of the primary site could lead to improved therapy. Veridex developed an optimized set of 10 gene markers, for a qRTPCR assay to identify tissue of origin of metastatic carcinoma in formalin-fixed, paraffin-embedded (FFPE) tissue samples (J Mol Diagn 8:320, 2006). The assay includes markers for 6 primary sites: lung, pancreas, colon, breast, ovary, and prostate. In this retrospective study, we evaluated the Veridex assay in patients with CUP. Methods: We obtained FFPE tissue from diagnostic biopsies on 69 CUP patients previously enrolled in empiric chemotherapy studies. The Veridex assay was performed as previously described. Assay results were correlated with clinical features, pathologic features, and response to treatment. Results: The Veridex assay yielded provisional diagnoses in 42 of 69 patients (61%): lung (15), pancreas (11), colon (12 ), ovary (4), breast (0), and prostate, (0 ). Most patients with diagnoses of lung and pancreas cancer had clinical and pathologic features compatible with these diagnoses; response rates to empiric chemotherapy (usually taxane/platinum-based) in patients with these diagnoses were 29% and 9%, respectively. The 12 patients with colon cancer diagnoses had predominantly intra-abdominal metastases (liver, peritoneum); response rate to therapy (usually taxane/platinum- based) was low (8%). The 4 patients with ovarian cancer had atypical clinical and pathologic features, and only 1 of 4 had PR to first-line taxane/platinum therapy. Conclusions: In this retrospective study, the Veridex 10-gene molecular assay was feasible and provided provisional diagnoses in a majority of patients with CUP. The diagnoses made using this assay (except ovarian cancer) were compatible with clinicopathologic features. The efficacy of cancer-specific treatment in patients diagnosed by this assay will be evaluated in prospective studies.

No significant financial relationships to disclose.
Triple-negative breast cancer. A clinicopathologic study of 113 cases and comparison with 55 cases with triple positive breast cancer: A matched-control analysis
Authors: El Weshi A. , Tulbah A. , Al Sayed A. , El Twegeri T. , AL Shabanah M. , AL Malik O. , Ajarim D.
Published: March 2020

21119

Background: Hormone receptor negative breast cancer (BC) is associated with poor outcome. BC patients (pts) with HER2/neu over expression (IHC 3+) are associated as well with poor prognosis. Aim: to further divide breast cancer to prognostic subgroups using interaction between these 2 prognostic factors. Methods: Between January 2002 and May 2004, the clincopathological features as well as treatment results of all non metastatic pts with triple negative BC (TNBC) and those with triple positive BC (TPBC) were reviewed. In all patients the three immunohistochemical markers, ER, PR and HER2/neu were available for review. Results: 113 TNBC pts and 55 TPBC were identified. In the TNBC group; median age was 42y, 76 % were premenopause, 21% with + family history (FH) and 6% were pregnant at diagnosis. Clinical stage (CS): I: 4%, II: 60%, III: 36%. Neo-adjuvant chemotherapy (NACT) was given to 29 %. Modified radical mastectomy (MRM) and breast conservative surgery (BCS) were performed in 49% and 51% respectively. Adjuvant CT (ACT) and radiotherapy (RT) were given to 85% and 84% of the pts. In the TPBC group, the median age was 43y, 71% were premenopause, 16% with + FH, and 2% were pregnant. The CS in the TPBC group was I: 4%, II: 65%, III: 31%. The distributions of NACT, MRM, BCS, ACT and RT in TPBC pts were 35%, 64%, 36%, 60% and 84% respectively. All TPBC pts received adjuvant hormonal therapy and non received adjuvant Herceptin. Relapse rate was 34% in TNBC and 20 % in TPBC (p = 0.04). At a median follow up of 40 months, 72% and 80% of the pts in the TNBC and TPBC groups are alive and disease free respectively. The 4y EFS and OS were 64% and 76% in TNBC and 74% and 89% in TPBC group (p = 0.04 and 0.008 respectively). Conclusions: TNBC is an aggressive neoplasm and has a poor outcome. More aggressive adjuvant chemotherapy and possible biological therapy directed against EGFR, based on previous released information of the prevalence of EGFR in this particular phenotype, should be considered in this group of patients.

No significant financial relationships to disclose.