bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of genotyping of gastrointestinal stromal tumor in Japan
Authors: Takahashi T. , Nishida T. , Sakurai S. , Kanda T. , Sawaki A. , Wada R. , Hasegawa T. , Hirota S.
Published: August 2020

e21502

Background: Most gastrointestinal stromal tumors (GIST) have activating mutations in the KIT or PDGFRA gene. Genotyping of GIST is important in Dx and Tx of GIST. Methods of genotyping using genomic DNA extracted from paraffin-embedded specimens are diverse and not standardized. We did validation study of genotyping using special reference to sequencing data obtained from cDNA from fresh GIST samples. Methods: Three DNA extraction methods (QIAamp, DEXPAT, or original) and four PCR methods (Ex Taq, AmpliTaq condition-1, AmpliTaq condition-2, or QIAGEN Tag) were compared using 20 paraffin-embedded specimens with special reference to sequencing data obtained from cDNA from corresponding 20 fresh GIST samples. After DNA extraction, KIT exon 9, 11, 13 and 17, and PDGFRA exon 12 and 18 were amplified by each PCR method using specific primers and directly sequenced. Results: In evaluation of PCR method, the protocol with Ex Taq showed 100% amplication of DNA and sequence agreement, the protocol with QIAGEN Tag 99%, and the protocol with AmpliTaq condition-2 86% agreement, and the protocol with AmpliTaq condition-1 showed much less amplication and higher disagreement. For the DNA extraction, the protocol with QIAamp showed best DNA extraction and its DNA sequence data were consistent with reference sequence in 98%, DNA sequence obtained using DEXPAT showed 33% consistency, and 89% of DNA sequence data obtained from an original method was agreed with reference data. Some modifications improved DNA amplication but inconsistent sequence data also increased probably due to miss-PCR. Conclusions: Each DNA extraction method had different quantity of DNA and four PCR methods showed different quality. Using this validation study, a standard genotyping method in Japan was established.

No significant financial relationships to disclose.
A phase II study of imatinib mesylate as adjuvant treatment for curatively resected high-risk localized gastrointestinal stromal tumors
Authors: Kang B. , Lee J. , Ryu M. , Im S. , Park S. , Kang W. , Kim T. , Oh D. , Jung K. , Kang Y.
Published: August 2020

e21515

Background: In our previous study, the presence of c-kit mutation as well as tumor size and mitotic count was an independent poor risk factor for relapse after curative resection of primary localized GIST. The patients with all the 3 poor risk factors had only 30% of 2 year relapse free survival rate (RFSR). (Kim, et al. Clin Cancer Res, 2004) It is also well known that c-kit exon 11 mutant GISTs respond better to imatinib treatment than the other mutant or wild type GISTs. Therefore, the patients who have primary localized GISTs with large size, high mitotic count, and c-kit exon 11 mutation may be the best candidate of adjuvant imatinib treatment. In this phase II study, we have evaluated the efficacy and safety of adjuvant imatinib for this patient group. Methods: Patients who underwent complete resection of a primary GIST with 1) c-kit exon 11 mutation, and 2) ≥10 mitoses/50 HPF, or tumor size ≥10 cm, or ≥5 mitoses/50 HPF and tumor size ≥5 cm were eligible. Patients received imatinib 400mg p.o. daily until recurrence of disease, intolerable toxicities, or for 2 years. The primary end point was relapse-free survival (RFS). Results: A total of 47 patients from 4 centers in Korea were enrolled. The median age was 57.0 years. Stomach was the most common primary site (n=31). Median primary tumor size was 7.5cm and median mitoses index was 12/50 HPF. With a median follow-up of 26.9 months, the median RFS and overall survival (OS) have not yet been reached. RFSR was 97.7% at 1-year and 92.7% at 2-years. Six relapses (12.8%) were documented among the 47 patients. The treatments were generally well tolerated. Grade 3–4 toxicities included neutropenia 27.7%, rash 8.5%, constipation 4.3%, anorexia 2.1%, vomiting 2.1%, and pruritis 2.1%. There were no episode of febrile neutropenia and treatment-related death. Conclusions: Postoperative adjuvant imatinib for 2 years were safe and could prolong the RFS in patients with high-risk of recurrence following complete surgical resection of the primary GIST. However, it remains unknown if this benefit in RFS can be translated into OS benefit. So, further follow-up is needed with comparison of OS with historical controls who could be use imatinib after recurrence.

[Table: see text]
Breast sarcomas: Is now the time to change our mind?
Authors: Galaychuk I.
Published: August 2020

e21524

Background: Primary breast sarcoma usually was considered a rare malignant disease [Adem et al., 2004]. The aim of this study is to show the incidence of breast sarcoma in the region with female population of about 600,000. Methods: Retrospective comparative analysis of breast sarcoma and breast cancer incidence in female of Ternopil region for 2000 - 2008. Descriptive statistical method was used. Results: The total of 101 cases of breast sarcoma was diagnosed in female of 39 - 76 years of age. Every year about 11 (6–18) primary breast sarcomas and 256 (233–279) patients with primary breast cancer were detected. The incidence rate of breast sarcoma was 1.14 per 100,000 women in 2000, 2.94 (in 2001), 1.48 (2002), 0.99 (2003), 2.16 (2004), 1.67 (2005), 2.53 (2006), 2.03 (2007) and 1.87/100,000 in 2008. The incidence of breast cancer was ranged from 37.3 to 46.4 per 100,000 of female population. Breast sarcoma consisted of 2.5% of the breast malignancies in 2000, 6.5% (in 2001), 3.3% (2002), 2.6% (2003), 5.4% (2004), 4.1% (2005), 5.9% (2006), 4.4% (2007) and 4.7% in 2008. Pathohistologic examination revealed angiosarcoma in 92.1% of cases. Thus, our findings are significantly different than data of other authors: f.e., the incidence of primary breast sarcoma is at 45 new cases per 10 million women [Confavreux et al., 2003], or 17 new cases per million women [Moore and Kinne, 1996], and breast sarcoma accounting for 0.0006% of breast malignancies which were diagnosed in 1910–2000 [Adem, 2004]. Taking into account all historical and recent data we can suggest beginning of biological changing in correlation between breast malignant tumors–carcinoma and sarcoma. Conclusions: Our statistical finding shows that incidence of breast sarcoma is 1.87 (0.99–2.94) per of 100,000 female population, and now sarcomas consist of 4.4% (2.5–6.5%) of all malignant breast tumors. Angiosarcoma is a most common (92.1%) morphological type of breast sarcomas.

No significant financial relationships to disclose.
In vitro sequence-dependent interaction between paclitaxel and gefitinib in human lung cancer cell lines
Authors: Cheng H. , Wu Y. , An S. , Dong S. , Chen H. , Zhang X. , Guo A.
Published: August 2020

e22025

Background: Cytotoxicity chemotherapy has been standard first line treatment for advanced NSCLC. Clinical trials comparing first line EGFR TKI therapy over cytotoxicity chemotherapy are under investigation in phase III trials. The aim of this study is to test the hypothesis that paclitaxel followed by gefitinib would be superior to the opposite order in EGFR TKI resistant cell lines because of cell signaling pathway and cell cycle interaction. Methods: we have used EGFR-TKI resistant human lung cancer cell lines A549, H1975 and H1650 as an in vitro model for defining the differential effects of opposite sequence of combination of cytotoxic drug and anti- EGFR agents on cell growth, signaling pathway, cell cycle distribution and induction of apoptosis. Results: Paclitaxel 24 hours followed by gefitinib 72 hours in A549, H1975 and H1650 cells produced synergistic effects, while the reverse sequence produced antagonistic effects. Exposure to paclitaxel resulted in an increased pEGFR and pAKT level, this increase of phosphorylation can be inhibited by the following gefitinib exposure, while the reverse sequence resulted in no change in EGFR and AKT phosphorylation. We confirmed that gefitinib arrested the cells in G1, paclitaxel arrested cells in S phase. The sequence of paclitaxel followed by gefitinib cause cells arrested in G1, while the reverse sequence cause cells arrested in S and G2 phase. Conclusions: These findings suggest that the sequence of paclitaxel followed by gefitinib may be superior to the reverse sequence in gefitinib resistant NSCLC, and support the investigation of these sequential treatment in the clinical setting. This work was supported by the grants from the National Natural Science Foundation of China (No. 30772531).

No significant financial relationships to disclose.
MicroRNA for circulating tumor cells detection in breast cancer: In silico and in vitro analysis
Authors: Medina Villaamil V. , Blanco Calvo M. , Díaz Prado S. M. , Antolín Novoa S. , Calvo Martínez L. , Santamarina Caínzos I. , Haz Conde M. , Antón Aparicio L. M. , Valladares-Ayerbes M.
Published: August 2020

e22027

Background: Detection of circulating tumor cells (CTC) may provide diagnostic and prognostic information in breast cancer (bc) patients. Deregulation of microRNAs (miRNAs) is frequent in tumors, including breast cancer. It is suggested miRNAs play a role in tumor progression. We hypothesized that miRNAs may constitute a promising new class of cancer biomarkers for CTC detection. Our objective has been to identify miRNAs potentially useful for CTC detection. Methods: Phase I preclinical study was performed by means of computational tools for miRNAs profiling including MIRGATOR, MIRBASE, SMIRNAdb, GeneHUB-GEPIS, MICRORNA.ORG, and MIRNAMAP. In silico data were used to identify and prioritize miRNAs highly expressed in breast cancer, but absent in hematopoietic-derived sources. Selected miRNAs were evaluated by means of qRT-PCR in breast cancer and Hematopoietic cell lines, normal blood, and blood from breast cancer patients. Results: Computational tools identify a set of miRNAs highly expressed in breast cancer sources in relation to hematopoietic samples. Among these were miR-141, miR-200c, miR-196a, miR-203, miR-200a, miR-200b, miR-32, miR-375, miR-31, miR-193a, and miR-205. For instance, relative expression of miR-32 was 104 higher in bc cell lines (N= 5) than in normal PB (N = 19) using qRT-PCR. In a preliminary analysis of PB from bc patients (stage IV) higher relative expression levels for selected miRNAs were found comparing with age-matched controls’ blood. Conclusions: Our results suggest that miRNA bioinformatic approach is a useful high-throughput method to select bc-associated miRNAS. The selected miRNAs should be further evaluated for their potential as markers for CTC detection. We next investigate if blood miRNA profile could predict tumor progression and survival. Supported by grants PI06/1541 and PI07/0477 from Fondo de Investigaciones Sanitarias (FIS), Instituto de Salud Carlos III.

No significant financial relationships to disclose.
Localization of erythropoietin receptor mRNA in primary breast tumors by laser capture microdissection
Authors: Miller C. P. , Loeb K. , Edlefsen K. , Urban N. , Blau C. A.
Published: August 2020

e22036

Background: Adverse effects of erythropoiesis stimulating agents on tumor progression and/or survival were observed in recent Phase III clinical trials, however, mechanisms are not understood. Whether tumor erythropoietin receptor (EpoR) expression is associated with erythropoietin-dependent tumor progression remains unclear, owing in part to the lack of specificity of commercial antibodies for protein detection. To overcome issues of protein detection, we previously optimized a quantitative RTPCR approach for reliable measurements of low level EpoR mRNA in primary tumor samples, and observed a 30-fold range of EpoR expression among panels of both breast and head and neck cancer samples. Methods: To test whether EpoR mRNA is expressed in tumor epithelial cells, we performed laser capture microdissection to fractionate 3 breast tumors into tumor epithelial enriched vs. depleted fractions. In each fraction, lineage marker and EpoR mRNA expression was normalized to 3 endogenous control genes. Results: Vascular endothelial markers in tumor epithelial-enriched fractions were reduced by an average of 15.3-fold (vascular endothelial cadherin) and 18.5-fold (platelet cell adhesion molecule 1) while the stromal marker vimentin was reduced by 14.7-fold. EpoR expression was enriched by 4.9-, 7.2-, and 1.1-fold in epithelial enriched fractions. Conclusions: Despite the depletion of endothelial cells, EpoR mRNA was at the same level or enriched in tumor epithelial fractions. These results demonstrate that malignant epithelial cells are a major source of EpoR mRNA in primary tumors. Applying this approach to additional breast cancer specimens as well as other cancer types will improve our understanding of erythropoietin-associated tumor progression.

[Table: see text]
APC germ-line allele-specific expression in familial adenomatous polyposis
Authors: Castellsague E. , González S. , Blanco I. , Guinó E. , Lázaro C. , Gruber S. , Capella G.
Published: August 2020

e22037

Background: About 13% of Familial Adenomatous Polyposis (FAP) families and 70% of Attenuated FAP families remain with unknown molecular pathogenic cause after APC and MYH mutational analyses. Also, mutations can affect specific allele expression (ASE) at the germline level. The aim of the study was to determine the presence of germline ASE in the APC gene in FAP and AFP with and without detectable APC or MYH mutations. Methods: Germline RNA from fresh frozen and/or cultured lymphocytes of 17 APC/MYH-negative Polyposis (7 FAP, 10 AFAP) families (21 individuals) and 35 APC-mutated Polyposis (30 FAP, 5 AFAP) families (60 individuals) was analyzed. Fourteen controls were also studied. ASE was investigated by single nucleotide primer extension (SNuPE) of rs2229992 APC coding SNP. Results: In controls ASE was 1.04± 0.3. We found that 17% (3 of 17) APC/MYH(-) FAP and AFAP families showed ASE (range=1.17–1.39) and ASE co-segregated with disease. ASE was more intense in short-cultured lymphocytes except for two cases and completely reversed by puromycin treatment. Eleven of 35 (31%) APC-FAP/AFAP harbored ASE (range=1.20–7.76), and the mutant allele was underexpressed in each case. ASE was restricted to splicing (4 families), nonsense (3 families) and frameshift (3 families) mutations outside of exon 15. Puromycin reversed ASE in all cases analyzed. Conclusions: APC ASE is present in a significant proportion (17%) of APC/MYH(-) FAP or AFAP. ASE, due to nonsense-mediated decay (NMD), is present in APC-FAP and is associated with specific mutation location, similar to reports for other hereditary syndromes.

No significant financial relationships to disclose.
Analysis of expression of TGF-β1 receptor (ALK-1) in normal and tumor tissues by tissue microarrays
Authors: Erbersdobler A. , Simon R. , Hellwinkel O. J. , Bokemeyer C. , Sauter G. , Hu-Lowe D. , Levin W. , Gallo-Stampino C. , Fiedler W.
Published: August 2020

e22044

Background: TGF-β1 is an important angiogenic factor involved in different aspects of angiogenesis and vessel maintenance. TGF-β1 receptors consist of class I, class II and accessory receptors. Activin-like kinase I (ALK-1) is a class I TGF- β1 receptor which is almost exclusively expressed on endothelial cells. Hereditary hemorrhagic telangiectasia (HHT) is caused by mutations in the ALK-1 gene. Knock-out mice for ALK-1 die during gestation due to vascular malformation. ALK-1 may thus represent an attractive target for anti-angiogenic therapy. A therapeutic monoclonal antibody (PF-03446962) against ALK-1 has been developed which is currently in phase I testing. Here we describe target validation in normal and tumor tissue using tissue micro-arrays (TMAs). Methods: Normal tissue arrays comprised of 609 individual histological samples representing 76 human tissues. Multi-tumor arrays consisted of 3923 individual tumor samples from 87 tumor types. Consecutive sections of tissue arrays were stained for ALK-1 and CD31. CD31 immunohistology (IHC) was performed to ensure presence of vessels within tissue samples. ALK-1 staining was graded 0=absent, 1=weak, 2= moderate and 3=strong by an experienced pathologist blinded to tissue type. Results: ALK-1 staining of vessels in normal tissues was generally weak (mostly grade 1 and rarely grade 2) and was detectable in lymphatic tissues including tonsil, lymph nodes, thymus and spleen, lung, the entire GI tract including parotid, submandibular and sublingual glands as well as pancreas. ALK-1 positive vessels were also found within the female genital tract including placenta, uterus and ovary. In human tumor vessels ALK-1 expression showed high variability between tumor types. The highest ALK-1 expression rate was found in lung cancer (NSCLC 49%, SCLC 83%), neuroendocrine pancreas tumor (71%), colon cancer (50%), chondrosarcoma 50%, angiosarcoma 40% and NHL (44%). Conclusions: TMAs are an excellent tool to verify target expression in normal and tumor tissues. Results may be used to predict side effects of targeted therapy and to direct phase II testing of innovative agents in patients likely to respond.

[Table: see text]
Treatment of VHL patients with sunitinib: Clinical observations and translational studies
Authors: Matin S. F. , McCutcheon I. E. , Gombos D. S. , Waguespack S. G. , Wen S. , Smith L. A. , Zhang Y. , Davis D. W. , Fuller G. , Jonasch E.
Published: August 2020

e22047

Background: Von Hippel Lindau (VHL) disease induces vascular lesions in multiple organs. Surgical intervention is the treatment of choice. Effective, safe systemic therapy will greatly improve the quality of life of these individuals. Methods: Patients with genetically confirmed VHL were enrolled on an IRB approved trial. Eligibility criteria included retinal angiomas, hemangioblastomas (HBs) measuring at least 5mm, renal cell carcinoma (RCC) 1 to 3 cm and pancreatic neuroendocrine tumors (NETs) 1 to 3 cm. Patients received sunitinib 50mg/day for 28 days/14 days off for four cycles. Scans were performed at baseline, and after cycles two and four. A separate set of 20 formalin-fixed paraffin embedded HBs and 20 RCCs were used for laser scanning cytometry (LSC) analysis of total and phospho vascular endothelial growth factor receptor (pVEGFR) and phospho platelet derived growth factor receptor (pPDGFR) levels in tumor endothelium. Results: Twelve patients were evaluable for response and toxicity. Eight had RCCs, nine had CNS lesions, and three had pancreatic NETs. Tumor size reduction was seen in 16/19 evaluable RCC lesions (95%CI 0.60, 0.97), 3/5 NETs (95%CI 0.15, 0.95) and 6/19 HBs (95%CI 0.13, 0.57). Two patients came off study for treatment related side effects, one patient progressed on study, and two patients did not complete the scheduled course of therapy. LSC analysis demonstrated significantly lower phospho VEGFR levels in HBs when compared with RCC. The mean (SD) of pVEGFR levels in log-transformation were 11.27 (0.49) and 11.75 (0.37) for HBs and RCC respectively (p = 0.003). The mean (SD) of pVEGFR to VEGFR ratio was .21 (0.12) versus 0.37 (0.43), for HBs and RCC respsectively (p = 0.043). Conclusions: Sunitinib treatment of patients with VHL resulted in consistent decrease of RCC and NET tumor size. The discrepant response to sunitinib in RCC and HBs may be explained by differential dependence on VEGFR activation in tumor endothelium.

[Table: see text]
Expression of stem cell markers in circulating melanoma cells
Authors: Fusi A. , Busse A. , Ochsenreither S. , Rietz A. , Keilholz U.
Published: August 2020

e22056

Background: Within circulating tumor cells there may be a subset of cell with stem cell (tumor initiating) characteristics able to develop distant metastasis. Several markers including nestin and CD133 have been found to be possible candidates to identify such a kind of subpopulation in other experimental models. We evaluated the presence of melanoma cells bearing stem cell phenotype in the bloodstream of patients with cutaneous or uveal melanoma after depletion of the leukocytes fraction. Methods: Between 50 and 100 ml of peripheral blood were collected from 12 melanoma patients with various tumor burden as well as three healthy volunteers. Blood samples were enriched for tumour cells by CD45 depletion of the leukocyte fraction using magnetic beads separation (EasySep, Stem Cell Technologies. Inc.). The remaining material was stained with antibodies for the markers Melan-A/Mart-1 (Dako) and HMB45 (Dako), CD133 (Miltenyi Biotec) and nestin (R&D System) and analysed by flow cytometry (BD FACSCalibur). Ten ml of blood were further processed and CD133, nestin, Melan-A/Mart-1 transcripts were quantified by Real Time RT-PCR (LightCycler, Roche Diagnostic). Results: CD45-depleted fractions in healthy controls were negative for melanoma markers. Melan-A/Mart-1 and/or HMB45 positive cells were detectable in 11 out of 12 melanoma patients. The absolute number of melanoma cells identified ranged from 6 to 176 per 10 ml of blood. Nestin expressing cells were more represented compared to CD133 expressing cells (median 27.4%, range 0.3% to 65.1% vs. median 9.3%, range 0.1% to 16.8%) within the melanoma fraction of cells positive for Melan-A/Mart-1 and/or HMB45. In one patient two different melanoma cell populations were detectable. The population of cells with lower expression of the melanoma markers showed at the same time higher expression of nestin and CD133 (5.9% vs. 1.3% and 10.2% vs. 6.7% respectively). Nestin results were in good accordance to the FACS data (nestin: r=0.55; CD133: r=0.23; Pearson test). Conclusions: The novel negative separation technique allows reliable isolation of melanoma cells from peripheral blood of patients with metastatic disease. A significant fraction of melanoma cells in peripheral blood bears a stem cell phenotype.

No significant financial relationships to disclose.
Predictors of survival in patients with metastatic breast cancer (MBC) treated with high-dose chemotherapy and autologous stem cell transplantation (HD-ASCT)
Authors: Von Drygalski A. , Tran T. B. , Messer K. , Pu M. , Corringham S. , Nelson C. , Ball E. D. , Ball E. D.
Published: August 2020

e22086

Background: Individualized care in MBC requires predictors of survival for tailored treatment. Although HD-ASCT has not resulted in improved overall survival (OS), retrospective analyses may identify patients who benefited. We reviewed records of all patients (n=96) in the bone marrow transplant registry at UCSD treated with HD-ASCT for MBC between 1989 and 2000. Methods: Age, race, stage at diagnosis, histology, estrogen receptor (ER) and menopausal status, body mass index (BMI) in kg/m2, time to transplant and death, sites of metastasis, disease status prior to and after transplant, and days in hospital were extracted. Brookmeyer & Crowley's 95% confidence intervals, Cox models for predictors of a time-to-event variable and Schoenfeld tests for proportional hazard assumptions were applied. Results: Median OS was 5.6 ys (CI 4.1–7.4) after initial diagnosis and 1.7 ys (CI 1.36–2.07) after transplant. OS after HD-ASCT at 12 ys was 8.2% and, although not statistically significant, 18.5% in ER- and 2.6% in ER+ patients, respectively. Stratified by ER status, stage at diagnosis was an independent predictor of OS. Patients with stage I at diagnosis were at lowest risk of death when compared to stage II-IV patients with HRs of 2.7 (II vs I CI 1.4–5.2), 4.6 (III vs I CI 2.1–10) and 17 (IV vs I CI 6.1- 47.8). Death risk was increased with BMI ≥ 30 (HR 3.1; CI 1.8–5.4), infiltrating lobular carcinoma (HR 2.5; CI 1.1–5.38) and visceral metastasis (HR 2.3; CI 1.3–4.1). Conclusions: The study highlights that ∼10% of patients experience ≥10 ys survival with HD- ASCT. Obesity, late stage at diagnosis, lobular infiltrating histology and visceral metastasis were independent negative predictors of OS. Surprisingly, ER- patients tended to live longer. Although survival was influenced by various disease characteristics, obesity was the only significant patient derived factor. How obesity negatively affects outcomes - whether affecting cancer growth or through associated morbidity - needs further study. These data may be useful stratification tools for future trials employing HD-ASCT as treatment modality in MBC.

No significant financial relationships to disclose.
18F-Fluorothymidine (FLT) as tumor response predictor for anti-epidermal growth factor receptor (EGFR) antibody in human lung cancer xenograft
Authors: Takeuchi S. , Zhao S. , Kuge Y. , Zhao Y. , Jin Y. , Hatano T. , Shimizu Y. , Kinoshita I. , Dosaka-Akita H. , Tamaki N.
Published: August 2020

e22088

Background: Specific EGFR inhibition therapies are currently promising and advanced in the clinical setting. It is revealed patients with a colorectal cancer bearing mutated K-ras do not benefit from cetuximab, a chimeric mouse-human antibody to EGFR. However, appropriate evaluation of the therapeutic effects has not been clarified yet, because of the lack of reliable tumor response predictors. Measurement of tumor proliferative activity by PET using FLT may serve to assess early response of the therapy. The purpose of this study is to determine whether 18F- FLT is useful to evaluate very early response to cetuximab treatment in human lung cancer cell line xenograft. Methods: Human tumor xenograft model was established in male BALB/c athymic mice by human lung cancer cell line NCI-H1975, which expresses L858R/T790M-EGFR. These mice were assigned to two groups (n=5, each group): control and cetuximab-treated groups. Cetuximab was intra-peritoneally given 1.0mg/body once on the first day. Biodistribution of 3H-FLT (%Inject Dose/g/kg) were studied two days after the treatment. Immunohistochemical staining of EGFR, phosphorylated EGFR (pEGFR), and nuclear antigen Ki-67 as a tumor cell proliferation marker in tumor tissues were also studied to assess early effects of cetuximab. Results: FLT uptake was high in tumors compared with any other organs. Two days after the cetuximab therapy, the mean value of FLT uptake in tumor was significantly decreased to 55% of control value (0.38 ± 0.03 in control group vs. 0.21 ± 0.07 in cetuximab-treated group; p < 0.05). There were no statistical differences in the tumor volume and body weight of mice between each group. High expression of EGFR and Ki-67 were observed in this xenograft. As compared with control, Ki-67 expression was remarkably decreased in cetuximab-treated group, while there were no definite changes in expression of EGFR and pEGFR. Conclusions: In this animal model, FLT uptake and Ki-67 expression were decreased in cetuximab- treated group just two days after the treatment start. These results indicate FLT-PET can be a useful predictor to evaluate very early response to molecular targeted medicine such as cetuximab before significant change of tumor size.

No significant financial relationships to disclose.
Does hormone receptor (HR) positivity affect the prognosis in breast cancers with human epidermal growth factor receptor 2 (HER2) overexpression?
Authors: Lee Y. , Sohn J. , Park B. , Chung H. , Suh C. , Kim S. , Koo J. , Kim J. , Choi H. , Kim Y.
Published: August 2020

e22091

Background: Biologically, there is an unclear issue about the role of HR positivity in HER2 positive breast cancer. These HER2(+)/ HR(+) pts were grouped into luminal B type apart from HER2(+)/ HR(-) pts in molecular profiling. However, from the clinical point of view, these pts have been categorized and been treated as either the only HER2(+) disease regardless of HR status or vice versa. Thus, we investigated the impact of HR status on clinical outcomes in HER2-overexpressed breast cancers. Methods: We retrospectively reviewed medical charts of HER2-positive breast cancer pts who underwent curative surgical resection from 1996 to 2001 in the Severance hospital, Korea. Demographic comparisons were performed by Chi-square tests. Tumor size, nodal stage, TNM stage, HR status, and adjuvant tamoxifen use were included in the Cox proportional hazards model. Results: Among the total 174 HER2-positive pts, HR (n=93) was positive in 53.5% (n = 93) and HR-positive tumors were more likely to be premenopausal (73% v 52%; P=0.01) and well- differentiated (grade 1or 2; 77% v 62%; P=0.04). There were no significant differences according to HR status in terms of tumor size, nodal stage, TNM stage, operation methods, and chemotherapy regimen. In these HER2-positive pts, the 5-year disease free survival (DFS) was longer in HR(+) pts than in HR(-) pts (DFS; 82.9% v 61.5%; P= 0.01). In a subset analysis, the 5-year DFS of HER2(+)/ER(+) pts without adjuvant tamoxifen (n=26) was not different from that of HER2(+)/ ER(-) pts (DFS; 57.7% v 61.5%; P= 0.32). However, the 5-year DFS of HER2(+)/ ER(+) pts with adjuvant tamoxifen was significantly prolonged compared with that of HER2(+)/ ER(-) pts (DFS; 91.5% v 61.5%; P< 0.001). In a multivariate analysis of DFS, tumor size and adjuvant tamoxifen use significantly affected DFS with an adjusted hazard ratio of 2.56 (95% CI, 1.2–4.9; P= 0.01) and 6.58 (95% CI, 2.8–20.3; P< 0.001), respectively. Conclusions: In an analysis of HER2-overexpressed breast cancer, the presence of HR itself did not affect the prognosis. However, most of the survival benefit seems to be driven from adjuvant tamoxifen therapy not the HR status itself.

No significant financial relationships to disclose.
The role of EGFR MAbs C225 in breast cancer stem cells
Authors: Shi Y. , Zhang F. , Cao M. , Zhang Y. , Xu Y. , Huang M. , Wang H. , Ma Y.
Published: August 2020

e22093

Background: The epithermal growth factor receptor(EGFR) signaling pathways has been implicated in self-renewal of breast cancer stem cells. It has been proved that the EGFR tyrosine kinase inhibitor-gefitinib significantly decreased the mammosphere- forming efficiency(MFE) in ductal carcinoma in situ (DCIS) derived cancer cells. We investigated the response of breast cancer stem cells in breast cancer cell line MCF-7 to EGFR MAbs C225(cetuximab). Methods: Cells were used for nonadherent (i.e.,mammosphere) culture and were divided into four groups according to the different culture medium whether or not including exogenous EGF and MAbs C225. MFE was calculated as the number of mammospheres (≥60um) and was expressed as a percentage. We Compared the percentage of CD44+/CD24- cells from the four groups, and measured the levels of the Wnt and Notch-3 gene expression of the four groups. Results: There was no significant difference of MFE between groups. In the presence of exogenous EGF, MAbs C225 significantly decreased the MFE and the percentage of CD44+/CD24- cells of mammospheres compared with exogenous EGF alone(0.7% versus 1.5%, P < 0.001 and 4% versus 11%P < 0.001;respectively). In the absence of exogenous EGF, MAbs C225 aslo statistically decreased the MFE and the percentage of CD44+/CD24- cells of mammospheres compared with that of lacking exogenous EGF and MAbs C225(0.53% versus 1.3%,P = 0.003 and 9% versus 3%, P < 0.01;respectively). And we observed that the Notch-3 gene expression was compensativly increased in the two groups of addition of MAb C225. No significant differences of Wnt gene expression were observed between groups. Conclusions: EGFR MAbs C225 could disrupt mammoshpere formation and decrease the percentage of CD44+/CD24- cells of mammoshpere cells. The expression of Notch-3 is increased with the EGFR antagonistics in breast cancer stem cells.

No significant financial relationships to disclose.
Molecular analysis of tamoxifen-resistant pathways in breast cancer cell-lines and breast cancer tissues
Authors: Yeoh C. , Chelala C.
Published: August 2020

e22094

Background: Oestrogen receptor positive (ER+) breast cancer (BC) is heterogeneous with regard to their clinical behaviour and response. The ER and Progesterone receptors (PgR) are currently the best predictors of response to the anti-oestrogen agent tamoxifen, yet up to 30–40% of ER+ breast cancers will relapse despite tamoxifen treatment. New prognostic biomarkers and further biological understanding of tamoxifen resistance is required. There has been an explosion of greater understanding since the arrival of new cutting-edge genomic profiling technology. Methods: RNA from the BC cell lines and frozen tissue was extracted and qualified using Qiagen Mini Kit and BioAgilent respectivelly. Probe preparation and scanning of the chips were carried out according to Affymetrix protocol. The normalisation and analysis of data (Limma) was performed using Bioconductor software. RT- PCR probes were purchased from Applied Biosystem and performed on ABI7500 instrument. The IHC-P was done using Ventana. TMA prepared with TMABooster, Alphelys (Plaisir, France). Results: Gene expression changes were studied in the TR breast cell lines MCF7 (tenovus), T47D and ZR by using the Affymetrix Hu133 plus 2.0 platform. Common genes of significant expression from the 3 TR cell lines were validated by real-time PCR (RT-PCR) and immuno-histochemistry (IHC-P) staining. 7 (MAGEA2, AKR1C3, VGLL1, THRAP5, GREB1, PDZK1, MYBL1, HnRNPA2B1, PGR) out of the 9 chosen genes were successfully validated. Proliferation studies on MageA2 overexpressing clones were performed. Stable overexpression of MageA2 in T47D and MCF-7 confers a proliferation advantage in tamoxifen-containing media as shown in proliferation and cell cycle analysis. MageA2 has been shown to alter the p53 pathway. The expression of KAP1, which interacts with p53 and inactivates it, was enhanced by MageA2 overexpression. In addition, overexpression of MageA2 is seen to increases histone deacetylase (HDAC3), and p53 expression as seen by immunoblotting. Validation of MageA2 on TMA of 100 patients is underway. Conclusions: MageA2 is a possible selective molecular target for the reversal of tamoxifen resistance.

No significant financial relationships to disclose.
Triple-negative breast cancer in Hong Kong Chinese patients
Authors: Tsang J. , Lai T. L. , Lau D. H. , Au G. K. , Chua D. T.
Published: August 2020

e22127

Background: There is increasing data showing that breast cancer is a heterogeneous disease which should be assessed separately in different populations, as it differs substantially between Chinese and Caucasian women. Triple-negative breast tumours which are negative for ER, PR and HER-2 neu receptors are associated with younger age at presentation, tumour of higher grade with larger size and a poorer prognosis. There is recent suggestion that the prognostic outlook of Chinese triple-negative breast cancers might be somewhat different from those in the Western population, but few studies have attempted to understand the role of ethnic factor in the triple-negative entity. Methods: We conducted a preliminary retrospective comparison of 170 Hong Kong Chinese primary breast cancer patients seen as new cases during January 2004 and December 2004 in a teaching hospital. Clinico-pathological features of triple-negative tumours were compared to their non-triple-negative counterpart. Results: Triple negative breast cancer accounted for 12.4% of all breast cancer patients seen in the year of 2004 (n = 21). It is associated with more cancers with grade 3 tumour (68.4% vs 36.8%; p = 0.02) but there was no statistically difference between the age of presentation, tumour size, extensive intraductal component, lymph node status and rate of local relapse or metastasis after adjuvant therapy. Subset analysis further revealed that when triple negative breast cancer patients (n = 21) were compared to the HER-2 positive patients (n = 40) in the studied population, HER-2 positive patients were still associated with higher proportion of node positive disease (57.5% vs 30.0%; p = 0.04). Disease free survival and overall survival were not studied due to limited follow-up time. Conclusions: Our preliminary findings suggested that Hong Kong Chinese triple-negative breast cancers are associated with a more favourable outlook and might behave differently when compared to their Western counterpart. Further large-scale study of the ethnic factor with long-term follow-up is warranted.

No significant financial relationships to disclose.
Role of E1B protein-dificient oncolytic virus in breast cancer stem cells
Authors: Zhang F. , Ma Y. , Xu Y. , Huang M. , Song C. , Lin S. , Zhang Y.
Published: August 2020

e22131

Background: Cancer stem cells have been indicated in the initiation of tumors and are even found to be responsible for relapses after apparently curative therapies have been undertaken. In breast cancer, they may reside in the CD44+CD24−/low population. Oncolytic adenoviruses enter cells through infection and can kill both proliferating and quiescent cells. We investigated the role of E1B protein- dificient oncolytic adenovirus in breast cancer stem cells. Methods: MCF-7 cells were infected by E1B protein-dificient oncolytic adenovirus as infected group (MOI=100) and cultured routinely as control group simultaneously. The proportion of CD44+CD24- cells was assessed by flow cytometry (FCM) in two groups respectively. Meanwhile, mammosphere culture was done in two groups' cells to observe the size and number of mammospheres, calculate the mammosphere- forming efficiency (MFE). The proportion of CD44+CD24- cells in two groups' mammospheres was assessed by FCM. Results: The percentages of CD24-,CD44+, CD44+CD24- in the infected gruop were 43.9%, 63.26%, 22.19%, respectively. While in the control group, the percentages were 6.74%, 88.30%, 2.30%. In the infected group, the time of mammosphere's formation was earlier, the volumes of mammospheres were bigger and the MFE was higher than the control group (1.26%:0.9%). In two mammospheres' groups, the proportion of CD44+CD24- cells in experiment group and control group was 38.08% and 23.35%, respectively. Conclusions: E1B protein-dificient oncolytic adenovirus can kill MCF-7 cells in short time, mainly breast cancer differentiated cells. It maybe promote the growth of the breast cancer stem cells. It maybe accelerate the speed of self-renewed and differentiation of the breast cancer stem cells.

No significant financial relationships to disclose.
Evaluation of plectin-1 immunohistochemical expression in human colon cancer tumor progression
Authors: Kannarkat G. J. , Harris T. , Kelly K. , Fracasso P. , Moskaluk C.
Published: August 2020

e22132

Background: Plectin-1 (PLEC1), a known scaffolding protein, was recently discovered to be upregulated and redistributed to the cell membrane in multiple cancers, thereby providing a molecular imaging biomarker for disease detection. The purpose of this study was to examine PLEC1 staining in the tumor progression of human colorectal carcinoma. Methods: A tissue microarray of colonic neoplastic progression was stained with antibody to PLEC1. A total PLEC1 immunohistochemical (IHC) staining score was obtained by multiplying the intensity of PLEC1 membrane staining, scored 1 to 3, by the percent of target cells stained, scored 1 (<25%), 2 ( 25%-75%) and 3 (>75%). Each specimen was grouped into low (0–2), intermediate (3–5) or high (6–9) expression. The mean IHC score and group distribution were obtained for each tissue type. Results: Normal colonic tissue showed the lowest membrane expression of PLEC1 with a mean IHC score of 2.24. Approximately 80% of cases were in the low group. Colonic adenomas had a slightly higher mean IHC score, 2.57, with more cases falling in the intermediate group. Invasive colorectal cancer as a whole had a mean IHC score of 3.96 with a third of cases falling into the high expression group. Inflamed colonic tissue (ulcerative colitis) stained the highest with a mean IHC score of 4.83 and all cases falling in the intermediate or high group. Conclusions: There is increasing membrane expression of PLEC1 in colonic epithelium through tumor progression. The observation of a difference in IHC staining of normal and malignant tissue requires larger confirmatory studies, but the redistribution of PLEC1 to the cell surface is a potential biomarker for molecular imaging of cancer and potential target for novel therapeutic agents. However, our finding of increased PLEC1 membrane staining in actively inflamed tissue may indicate an important caveat in the use and implementation of this biomarker in certain clinical situations.

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No significant financial relationships to disclose.
Prophylactic surgery and ovarian cancer treatment of women carriers of BRCA 1 gene mutation
Authors: Rzepka-Górska I. , Chudecka-Glaz A. , Górski B.
Published: August 2020

e22139

Backgrounds. Tree BRCA 1 mutations commonly present in Polish population (5382 insC, C61G, 4153 delA) have been implicated in familial clustering of mammary-ovarian cancer. Prophylactic bilateral adnexectomy reduce the risk of ovarian cancer of more then 80% and the risk of brest cancer to 53%. Methods. It is retrospective study. We enrolled 193 women carriers of BRCA 1 mutation. 161 had prophylactic adnexectomy and 32 were treated because of diagnosed ovarian cancer. All were treated at our Department of Gynecological Surgery and Gynecological Oncology of Adults and Adolescents between 1999–2008. We analysed the frequency of brest cancer in patients who were admitted to the hospital for prophylactic surgery and number of ovarian cancer diagnosed during prophylactic surgery. We analysed the response on standard treatment based on platinum analogs in carriers of BRCA 1 with ovarian cancer. It was also evaluated the incidence of primary peritoneal cancer in patients after prophylactic treatment. Results. The age of women at the time of prophylactic operation were between 41–60 y.o (mean 45 years old). We found, that 61 of patients had already diagnosed and were treated because of breast cancer (37.9%). During prophylactic surgery we diagnosed 10 cases of ovarian cancer, 2 cases of primary peritoneal cancer and 2 fallopian tubes cancer. These patients had no symptoms and were qualified to prophylactic adnexectomy. 32 women with ovarian cancer diagnosis had typical response to standard treatment, 70% were platinum sensitive and DFS was more then 12 months, 25 % had DFS between 6–12 months and 5% were platinum resistant. There were diagnosed only 2 cases of primary peritoneal cancer in patients after 31 and 61 months from prophylactic treatment. Conclusions. Separation the risk group - patients carriers BRCA 1 mutation let us to introduce effective prophylactic rules of conduct.

No significant financial relationships to disclose.
The reliability of HER2-status determination from core-needle-biopsies and surgical specimens: A comparison of two established test methods (IHC, CISH)
Authors: Wolf V. , Groβe R. , Erggelet J. , Holzhausen H. J. , Hauptmann S. , Thomssen C.
Published: August 2020

e22141

Background: A milestone of breast-cancer therapy was the discovery of HER-2 entailing special targeted therapy with improved prognosis. The HER-2-status is routinely assessed through immunohistochemistry (IHC; HercepTest) showing protein over-expression and is double-checked with in-situ-hybridisation (ISH) demonstrating gene amplification in equivocal cases. It is questioned whether these methods achieve identical results in core-needle-biopsies and in excisional tumor specimens. Methods: We performed a retrospective comparative study in order to address these questions. From 01/03–06/08 we collected the HercepTest results from both core-needle-biopsy and surgical specimen of 109 breast cancer patients in our institute and compared these to newly evaluated chromogenic ISH (CISH) results for both specimen types in order to assess the reliability of HER-2- diagnosis of both methodological approaches and of specimen type. Results: We found no significant difference in the HER- 2-status determined from either needle-biopsies or surgical specimens irrespective of the test used. For the overall comparison (218 specimens) of HercepTest and CISH we found only slight, non-significant deviations. Four cases were CISH-negative in spite of HercepTest scoring of 3+. Vice versa, five out of the total of 38 (17.4%) CISH-positives did not correspond to the HercepTest results of 0 or 1+. Conclusions: Though not significant, there is some inconsistency in the HER-2-determination depending on the test-method, leaving these cases equivocal. In accordance with the literature, we therefore recommend to at least double-check samples with 2+ in the HercepTest as it is the current standard. Our data support the use of core-needle-biopsy as a reliable tissue sample for HER-2-diagnosis.

[Table: see text]
Interactions between neutrophils and NSCLC cells in vitro effects on neutrophil inflammatory mediator generation and tumour cell proliferation
Authors: Hattar K. , Franz K. , Ludwig M. , Banat A. , Seeger W. , Grimminger F. , Sibelius U. , Grandel U.
Published: August 2020

e22148

Background: Growing evidence indicates that interactions between tumour cells and the inflammatory cellular microenvironment are crucial for tumour biology. In NSCLC, both bacterial infections and leukocytosis are associated with a poor prognosis. Against this background, we studied the effect of normal bronchial epithelial cells (B2B) and NSCLC cells (A549) on inflammatory neutrophil behaviour and quantified tumour cell proliferation in the presence of neutrophils. Methods: Isolated human neutrophils were co-cultured with B2B or A549 cells. Neutrophil respiratory burst was quantified by cytochrome C reduction and elastase activity was measured by the kinetics of substrate turnover. Proliferation of A549 cells was determined by MTS- assay. Results: Co-culturing human neutrophils with A549 cells stimulated neutrophil respiratory burst and degranulation in response to the bacterial tripeptide fMLP. In contrast, when co-cultured with B2B cells, these inflammatory neutrophil reactions were blunted. This could be attributed to release of NO and adenosine by the B2B cells. The increase of oxygen radical release and elastase degranulation in the presence of A549 cells was mediated by direct cell-to-cell contact. COX-2 dependent prostanoids were obviously involved in the activation of inflammatory neutrophil behaviour, as shown by the inhibitory capacity of the COX-2 specific inhibitor NS-398 in the co-culture system. Interestingly, activated neutrophils induced proliferation of NSCLC cells in a dose-dependent manner. Conclusions: The reciprocal interactions between neutrophils and NSCLC cells may enhance neutrophil-derived tissue destruction and tumour growth under inflammatory conditions.

No significant financial relationships to disclose.