bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of concurrent ovarian and endometrial carcinoma by copy-number analysis (CNA) and gene expression profiling (GEP)
Authors: Guirguis A. , Elishaev E. , Timms K. , Reid J. , Abkevich V. , Gutin A. , Launchbury J. , Zorn K. , Deloia D.
Published: March 2020

10546

Background: Coexistence of endometrioid carcinoma at both sites, the ovary and the endometrium, is between 10–30%. Current staging relies on clinical and pathologic assessment, which is not quantitative and highly subjective. The objective of this study was to evaluate archived tissues from patients with concurrent tumors for CNA and GEP, and determine if either or both of these techniques could be used to define the primary site of the endometroid carcinoma. Methods: A gynecologic pathologist confirmed that all specimens from both sites were of endometrioid histology; grades I/II. CNA was determined by extracting DNA from both disease and non-disease tissues for hybridization to the Affymetrix 500K SNP microarrays. Illumina HumanRef8 BeadChip microarrays were used throughout to obtain GEP for 7 endometrial and 7 ovarian primary stage I tumor samples. Differentially expressed genes between both groups created a corresponding discrimination scores (DS). The performance of the discrimination scores were evaluated by the Leave-One-Out method. In a similar fashion, a test set of 16 tumor samples from patients with concurrent tumors were profiled. The prediction of these tumors’ origin based on the DS were compared with pathological assessment and copy number analysis. Results: Concurrent tumors display more copy number changes than singular tumors of either tissue, with a greater fraction of the genome involved. Analysis of the non-affected tissue assists in identifying tumor-specific changes. We identified a set of 30 differentially expressed genes between both ovarian and endometrial tumors that discriminates a tumors’ origin with about 80% confidence. For patients with concurrent tumors, comparison of their origin based on GEP, CNA, and pathological assessment shows limited concordance. Conclusion: Shared somatic copy number changes in simultaneous endometrioid carcinomas of the ovary and endometrium indicate a common origin and hence metastasis. For these relatively common concurrent tumors, clinical inferences based on CNA appear more robust than those based on GEP, and more objective than pathological assessment.

No significant financial relationships to disclose.
Pathologic predictors of BRCA1/ BRCA2 (B1/B2) mutations in African-American (AA) women with early-onset breast cancer (BC)
Authors: Domchek S. M. , Simon M. S. , Hughes Halbert C. , Rebbeck T. R. , Nathanson K. L.
Published: March 2020

10549

Background: African-American (AA) women more frequently have “triple negative” (ER-, PR-, HER2-) breast cancer (BC) and such BC are more frequently associated with B1 mutations; however, the correlation of triple negative BC in the AA population and B1/2 mutations is not known. We were interested in examining the prevalence of B1/2 mutations in a cohort of AA with early-onset breast as well as the associated pathologic characteristics. Methods: 85 unselected AA women diagnosed with 88 BC at or prior to age 40 were enrolled on study and underwent BRCA1/2 genetic testing by full sequencing at Myriad Genetics following informed consent. MLPA screening for large genomic rearrangements also was performed. Pathology reports were obtained and examined. Results: Of the 85 women, 16 (19%) had either a B1 (11) or B2 (5) deleterious mutation, which were associated with 19 BC. 19/85 (22%) women had a variant of unknown significance (VUS); 3 had B1 VUS, 15 had B2 VUS (including 2 with B1 mutations) and 1 had both B1 and B2 VUS. No mutations were detected with MLPA. Of the 14 BC in 11 B1 mutation carriers, 12 were ER- (86%), 13 were PR- (93%) and 3/12 were HER2+ (25%). Of the 5 BC in B2 carriers, 4 were ER- (80%), 1/4 HER2+ (25%). Receptor status was available in 77 of 88 cases (88%). Overall 41 of 77 (53%) cancers were ER-. 15 of 40 women (38%) with any ER- BC had either a B1 (11) or B2 (4) mutation. In contrast, of the 36 ER+ BC, 2 had a B1 and one had a B2 mutation (8%) (p=0.01). Both B1 carriers with ER+ BC also had a contralateral ER- BC. 18 of 65 (27%) cancers were known to be HER2+, of which 4 of 18 (22%) were associated with B1 or B2 mutations (p=NS). Median age of first BC in B1/B2 mutation carriers was 32 vs. 37 in non-carriers. Conclusions: In unselected AA women with early onset BC, B1/2 mutations were common and predominantly found in association with ER- BC. B2 VUS were detected frequently. HER2 overexpression did not predict against B1/2 mutations.

No significant financial relationships to disclose.
Trastuzumab (T) plus capecitabine (C) in heavily pretreated patients (pts) with advanced breast cancer (ABC)
Authors: Bartsch R. , Wenzel C. , Altorjai G. , Pluschnig U. , Locker G. J. , Rudas M. , Mader R. M. , Zielinski C. C. , Steger G. G.
Published: March 2020

1055

Background: In pts with Her2 positive ABC, taxane or vinorelbine plus T are among the most widely applied options in the first line setting. We evaluated the efficacy and tolerability of TC in pts with Her2 positive ABC after anthracycline and docetaxel or vinorelbine failure. Methods: Forty consecutive pts (median age 57.5 years) were included. As of December 2006, all are evaluable for toxicity and 35 for response. C was administered at a daily dose of 2,500 mg for two consecutive weeks (w) every 3 w, with dose modifications if necessary. T was administered in 3 w cycles at a dose of 6 mg/kg bodyweight after a loading dose of 8 mg/kg. Time to progression (TTP) was defined as primary endpoint. Response was evaluated every three months (m) using UICC criteria. TTP and overall survival (OS) were estimated using the Kaplan-Meier product limit method. Differences in TTP for 2nd line and beyond 2nd line were analyzed with the log-rang test. Results: All pts had prior exposure to an anthracycline and at least one anti-microtuble agent (i.e. a taxane or vinorelbine). All had at least one earlier T containing treatment line for ABC. Median time of observation was 18.5 m. We observed a complete response in 2.9%, partial response in 20%, stable disease = 6 months in 48.6%, and progression in 28.6% of pts. OS was median 24 m (95% CI 20.3–27.7), and TTP 8 m (95% CI 5.8–10.1). No significant difference was found for 2nd and beyond 2nd line treatment. Diarrhoea (5%) and hand foot syndrome (16%) were the only treatment-related adverse events that occurred with grade 3 or 4 intensity. A dose reduction was necessary in 22.5%. Two pts developed brain metastases (BM) while on therapy, 6 had BM at time of treatment initiation, a further 5 developed BM during follow up. Of 6 pts with BM, 3 gained clinical benefit from treatment (one pt not yet evaluable). Conclusions: TC appears to be an effective and safe option as salvage therapy in a heavily pretreated population. TTP and response rates are similar to results from C plus lapatinib. Of note is the activity in pts with BM. Further, only 2 pts (5%) developed BM while on treatment. Therefore, a direct comparison of TC with C plus lapatinib or T plus lapatinib seems warranted.

No significant financial relationships to disclose.
Clinical significance of Epstein-Barr Virus DNA load detected pre- and post-radiotherapy in nasopharyngeal carcinoma patients
Authors: Hou X. , Zhang L. , Zhao C. , Lu L. , Han F. , Li S. , Huang P. , Huang H. , Guan Z.
Published: March 2020

10558

Background: Plasma Epstein-Barr virus DNA (EBV DNA) load has been shown to be clinically useful in the detection, monitoring, and prognostication of nasopharyngeal carcinoma (NPC). However, the clinical significance of EBV DNA load detected at different time points has not been addressed to our knowledge. In this study, we investigated whether pre- and post-treatment plasma EBV DNA load have different prognostic implications in NPC patients who were treated with radiotherapy. Methods: Plasma samples from 69 patients with primary NPC were collected before and after radiation treatment, and subjected to a real-time quantitative polymerase-chain- reaction assay of EBV DNA load. The pre-treatment primary tumor volume (GTVnx) calculated through CT images and/or MRI were documented. All patients were scheduled to follow up. Results: The pre-treatment plasma EBV DNA concentration was significantly associated with primary tumor volume (Spearman correlation coefficient=0.614; P=0.000). With a cutoff value of 20,000 copies/ml and 0 copies/ml respectively for pre-treatment and post-treatment plasma EBV DNA copy number, patients with lower EBV DNA concentrations had statistically preferable progression-free survival,metastasis-free survival and overall survival compared with those with higher EBV DNA concentrations. Cox regression analysis demonstrated that both pre-treatment EBV DNA load (P=0.050;RR=3.95) and post-treatment EBV DNA load (P=0.001;RR=11.74) were risk factors for metastasis-free survival. When further integrating pre-treatment with post-treatment concentration of EBV DNA, it was demonstrated that whether EBV DNA concentration could be dropped to 0 after treatment dominate the prognostic effect for metastasis-free survival (P=0.000). Conclusions: Pre- and post-treatment plasma EBV DNA have different clinical significance. Pre-treatment plasma EBV DNA is a reliable molecular marker reflecting primary tumor volume. While the clearance of circulating plasma EBV DNA after treatment is a good predictive marker of freedom from distant metastasis.

No significant financial relationships to disclose.
An investigational rapid RT-PCR assay for the detection of metastasis in sentinel lymph nodes shows improved performance over frozen section H&E: Analysis by primary tumor characteristics
Authors: Blumencranz P. , Deck K. B. , Whitworth P. W. , McCue P. , Reintgen D. S. , Beitsch P. , Chagpar A. S. , Julian T. B. , Mamounas M. , Simmons R.
Published: March 2020

10561

Background: In order for a RT-PCR test to accurately diagnose the metastatic status of sentinel lymph nodes the test must perform well across all primary tumor types. Methods: A prospective study was conducted at 11 clinical sites to evaluate a real- time RT-PCR assay (investigational GeneSearch™ Breast Lymph Node Assay, Veridex, LLC, Warren, NJ, USA) for the detection of sentinel lymph node metastasis in patients with invasive breast cancer. Detection limits were pre-set to detect only metastases that are clinically relevant (> 0.2 mm). Tumor information such as tumor stage, size, type and other molecular characteristics were collected. RT-PCR assay was then compared against permanent section H&E and IHC for final performance calculations. A total of 416 patients’ results were analyzed for overall assay performance: sensitivity 87.6% and specificity 94.2%. The assay was then evaluated within each primary tumor characteristics category. Results: Overall RT-PCR sensitivity is an improvement (p= 0.039) over that of current intra-operative methods (frozen section H&E), while specificity is similar (p=0.054). For the various tumor sub-types, in a matched dataset, the RT-PCR assay has up to 35.7% (Invasive Lobular) higher sensitivity compared to frozen section H&E. Conclusions: The data suggest that the RT-PCR assay performs well and has higher sensitivity than frozen section H&E regardless of primary tumor characteristics and, therefore, can be used to detect metastasis for all types of invasive breast cancer.

[Table: see text]

No significant financial relationships to disclose.
The effect of P53 gene status on the interaction of vorinostat (Suberoylanilide Hydroxamic Acid-SAHA) with carboplatin in non-small cell lung cancer (NSCLC) cell lines
Authors: Hershberger P. , Owonikoko T. K. , Ramalingam S. , Belani C. P.
Published: March 2020

10567

Background: Vorinostat, a Histone Deacetylase (HDAC) inhibitor is a novel targeted antineoplastic agent with promising activity when combined with carboplatin-paclitaxel against NSCLC. The exact molecular mechanism underlying its growth inhibitory and apoptotic effects is not well understood. We investigated the influence of p53 gene status on the interaction of vorinostat and carboplatin (a DNA targeting agent) in various NSCLC cell lines. Methods: NSCLC cells with wild type p53 (A549, 128.88T), mutant p53 (201T) and p53 null phenotype (Calu-1) were used. Cytotoxicity induced by serial dilution of carboplatin in the presence and absence of a fixed dose (1μM) of vorinostat was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. In a separate experiment, cells were also transiently transfected with a p21 promoter-luciferase reporter construct to assess p53 activation following a 24 h exposure to vorinostat, carboplatin, or vorinostat/Carboplatin combination. Luciferase activity was quantified by luminometry and corrected for total protein. Results: Vorinostat displayed single agent activity in each cell line, with greater growth inhibition observed in the p53 mutant and null cells. Synergistic interactions between carboplatin and vorinostat were observed in p53 wild type cells and the IC50 for carboplatin was reduced 3- to 5-fold. In contrast, the interaction between vorinostat and carboplatin was additive or less than additive in p53 mutant and p53 null cells. Vorinostat also increased expression of the p21 reporter construct in each of the cell lines. Conclusions: Vorinostat regulates p21 gene expression and elicits anti-tumor activity in NSCLC cells independent of their p53 status. Vorinostat potentiates carboplatin-induced cytotoxicity in NSCLC with wild type p53 but not p53 deficient cells, suggesting involvement of a p53 dependent pathway. The addition of vorinostat may allow for a reduction in standard dose of carboplatin with improvement in overall therapeutic index. A phase II/III clinical trial is in progress to evaluate vorinostat in combination with carboplatin-based regimen in advanced NSCLC. Support: CA099168–01, ASCO Foundation CDA

No significant financial relationships to disclose.
Expression and activation of mTOR in neuroendocrine tumors. Effects of mTOR inhibition by RAD001 upon growth, cell cycle regulation and signalling in neuroendocrine cell lines
Authors: Hörsch D. , Tielke S. , Schrader J.
Published: March 2020

10570

Background: Molecular pathogenesis of neuroendocrine tumors of the digestive tract is largely unknown. We examined paraffin sections of different types of neuroendocrine tumors for expression of activated kinases involved in regulation of growth and protection against apoptosis. In addition, we studied effects of a newly developed inhibitor of mTOR (RAD001) upon growth, cell cycle regulation and signaling in two different neuroendocrine cell lines. Methods: Immunohistochemistry with antibodies for native and activated signaling kinases was performed on paraffin sections. MTT assays were used to determine growth rates of neuroendocrine cell lines and FACS analysis was performed for cell cycle analysis. Immunoblotting revealed activation of kinases. Results: Extracellular activated kinase 1/2 (ERK1/2), cAMP regulated element binding protein (CREB), protein kinase B (PKB) and mTOR were expressed in the majority of insulinomas (n = 40) and serotonin-producing neuroendocrine tumors (carcinoids, n = 49). Phosphorylated forms of these kinases corresponding to their activated state were also detected in these tumors. Most neuroendocrine tumors expressed high levels of activated CREB, PKB and mTOR whereas phosphorylated ERK1/2 was present only a minority of dNETs. Inhibition of mTOR kinase by different concentrations of RAD001 induced growth arrest in insulinoma (INS-1) and human neuroendocrine serotonin-producing cells (BON) in concentrations between 10 and 100 nM. Interestingly, we observed pronounced synergism between octrotide and RAD001 upon growth inhibition in these cell lines. Growth inhibition by RAD001 corresponded to decreased S-phase and increased G0/G1 peak in cell cycle analysis. RAD001 inhibited effectively phosphorylation of mTOR with an IC50 of 10 nM. However, we observed only minor inhibition of PKB and CREB by RAD001 and partial inhibition of ERK signaling by RAD in concentrations between 100 nM and 1 μM. Conclusions: mTOR is expressed and activated in different neuroendocrine tumors and inhibition of mTOR by RAD001 induces growth inhibition, cell cycle arrest and decreased signaling by mTOR and ERK1/2 in neuroendcrine cell lines INS-1 and BON.

No significant financial relationships to disclose.
Overexpression of chemokine receptor CXCR4 in cancer specimens following neoadjuvant chemotherapy predicts outcome in patients with locally advanced breast cancer (LABC)
Authors: Holm N. T. , Byrnes K. , MacDonald M. , Abreo F. , Ampil F. , Sanders M. , Burton G. V. , Li B. , Chu Q.
Published: March 2020

10578

Background: Despite significant advances made in the treatment of locally advanced breast cancer (LABC) with neoadjuvant chemotherapy, a significant number of patients continue to die. A molecular predictor to identify those who are at an increased risk for relapse is sorely needed. CXCR4 is a chemokine receptor that has been linked to breast cancer invasion and metastasis. We postulate that CXCR4 overexpression levels in cancer specimens following neoadjuvant chemotherapy predict cancer outcome in patients with LABC. Methods: 54 patients with LABC were prospectively accrued and analyzed. All had neoadjuvant chemotherapy, followed by definitive surgical and adjuvant chemo-radiation therapy. Study homogeneity was maintained by standardized treatment, surveillance, and compliance protocols. A 1 cm 3 cancer from the surgical specimens of each patient was retrieved for analysis. CXCR4 levels were detected using Western blots and results were quantified against 1 μg of HeLa cells (positive controls). CXCR4 expression was defined as low (<6.6 fold) or high (= 6.6 fold). Primary endpoints were cancer recurrence and death. Statistical analysis performed included Kaplan-Meier survival analysis, log-rank test, and Cox proportional hazard model. Results: With a median follow-up of 30 months, patients whose tumors had high CXCR4 overexpression (= 6.6 fold) had a statistically significantly higher incidence of recurrence (p= 0.0009) and cancer-related death (p= 0.0168) than those in the low CXCR4 group (< 6.6 fold). After adjusting for tumor size, nodal status, ER, PR and HER-2 status, the relative risk for recurrence and death in the high CXCR4 group was 27.3-fold (p=0.001; 95% CI: 6.2 to 120.8) and 4.8-fold (p=0.0076; 95% CI: 1.5 to 15.0) higher than those in the low CXCR4 group, respectively. Conclusion: High CXCR4 overexpression in cancer specimens following neoadjuvant chemotherapy was highly predictive of cancer recurrence and cancer death in patients with LABC.

No significant financial relationships to disclose.
Vinflunine in combination with trastuzumab for treatment of HER2-positive metastatic breast cancer
Authors: Paridaens R. , Wildiers H. , Dalenc F. , Rixe O. , Cadic V. , Pinel M. , Roche H.
Published: March 2020

1058

Background: Vinflunine (VFL) is a microtubule inhibitor of the vinca alkaloid class with high activity in several tumour types especially in second line treatment of metastatic breast cancer (MBC). Over-expression of HER2 is observed in 20–25% of all breast cancers and is associated with a poor prognosis. The development of the anti-HER2 therapy with trastuzumab (T) allowed significant prolonging of the survival in HER2 positive MBC. The combination of these two compounds which are separately active in MBC warranted a combination study. Methods: This phase I was designed to determine the recommended dose (RD) of VFL in combination with T for treatment of first or second line MBC patients (pts), to assess its safety profile, and to determine its antitumour activity. Results: Thirty pts, median age 55 [33–74] years, WHO performance status 0/1: 18/12 pts , respectively were given VFL 280 mg/m2 (10 pts) or 320 mg/m2 (20 pts) day 1 every 3 weeks (w) in combination with T (loading dose 4 mg/kg and subsequently 2 mg/kg/w). Six pts were chemonaive and 5 pts received this combination in second line. At the first VFL dose (280 mg/m2), 0/6 pts experienced dose-limiting toxicity. The RD was established at 320 mg/m2 in the following 6 pts. All 30 pts are evaluable for safety. Haematological toxicities were: anaemia grade (G) 3: 0 and 1 pt; neutropenia G 3/4: 5/17 pts, febrile neutropenia 0/2 pts with VFL 280 and 320 mg/m2, respectively. G 3 non-haematological adverse events were only reported in the 20 pts receiving VFL 320 mg/m2: fatigue and myalgia: 5 pts; constipation: 4 pts; abdominal pain, neutropenic infection: 2 pts. ileus, hyponatremia, arthralgia: 1 pt. No significant cardiac events were observed. The response rates, 62.5% [95% confidence interval: 24.5–91.5] and 73.7% [95% confidence interval: 48.8–90.9] in pts receiving VFL at 280 mg/m2 and 320 mg/m2 respectively, were established by an external radiologist using RECIST in the first 27 evaluable patients. Conclusions: Vinflunine/trastuzumab combination is feasible with encouraging response rates and manageable toxicity in MBC pts. A phase III study comparing VFL + T versus paclitaxel + T has been initiated.

No significant financial relationships to disclose.
Stage III colon cancer prognosis prediction by gene expression profiling
Authors: Barrier A. , Boelle P. , Brault D. , Houry S. , Lacaine F. , Flahault A. , Dudoit S. , Lemoine A.
Published: March 2020

10590

Background and Aims: Postoperative chemotherapy has become part of the standard treatment for stage III colon cancer patients. Since approximately half of patients are cured by surgery alone, adjuvant chemotherapy should not be used in all patients. This study aimed to assess the possibility to use microarray-based gene expression profiles to predict the prognosis of stage III colon cancer patients. Material and Methods: Forty-two patients operated on for a stage III colon cancer were included in this study. Twenty-one patients have received an adjuvant chemotherapy, while the other 21 have received no treatment. Twenty patients have developed a liver metastasis, while the other 22 have remained disease-free for at least 5 years. Tumor mRNA samples were profiled using the Affymetrix HGU133A GeneChip. Two analyses were performed: one with the 42 patients, the other with the 21 patients who did not receive any adjuvant chemotherapy. For each analysis, patients were repeatedly and randomly divided into 10,000 training (TS) and validation sets (VS) of 10 different sizes. For each TS/VS split, a 30-gene prognosis predictor (PP), identified on the TS, was used to predict the prognosis of VS patients. Performances of a 15-gene PP and a 34-gene PP, proposed by another research team, were also assessed on the same TS and VS. Results: First analysis (42 patients). The 10,000 30-gene PP yielded the following average prognosis prediction performance measures: 73.8% accuracy, 74.6% sensitivity, and 73.0% specificity. Improvements in prognosis prediction were observed with increasing TS size. A total of 4,446 genes were included in the 10,000 PP. The 15-gene PP yielded a 69.7% accuracy; the 34-gene PP yielded a 71.2% accuracy. Second analysis (21 patients). The 10,000 30-gene PP yielded the following average prognosis prediction performance measures: 77.7% accuracy, 75.8% sensitivity, and 79.9% specificity. Improvements in prognosis prediction were observed with increasing TS size. A total of 5,478 genes were included in the 10,000 PP. The 15-gene PP yielded a 78.5% accuracy; the 34-gene PP yielded a 81.9% accuracy. Conclusion: Microarray gene expression profiling is able to predict the prognosis of stage III colon cancer patients and, thus, might be used for an appropriate use of adjuvant chemotherapy.

No significant financial relationships to disclose.
Elevated serum M-CSF level predicts reduced survival in metastatic breast cancer patients
Authors: Leitzel K. , Ettenberg S. , Walsh R. , Abraham J. , Modur V. , Braendle E. , Evans D. B. , Ali S. M. , Demers L. , Lipton A.
Published: March 2020

10591

Background: Macrophage colony stimulating factor (M-CSF) and its receptor (CSF-1R, the c-fms oncogene protein product) have been reported to be expressed in a variety of cancers, including breast cancer. The M-CSF produced by breast cancer cells and surrounding stromal cells increases osteoclast formation and maturation and enhances the expression of stromal RANK ligand, both of which increase osteolytic bone degradation. In this study we evaluated the predictive and prognostic potential of circulating M-CSF in metastatic breast cancer patients treated with hormone therapy. Methods: Using an M-CSF ELISA (R&D Systems, Minneapolis, MN), M-CSF concentration was determined in pretreatment sera from 204 metastatic breast cancer patients enrolled in a phase III 2nd-line hormone therapy trial of fadrozole vs. megace, and also in sera from 25 post-menopausal control female subjects. Results: The serum M-CSF level from the 25 healthy post-menopausal female control subjects had a mean ± SD of 835.6 ± 276.1 pg/ml (range 319.0 - 1,465.8 pg/ml). The upper limit of normal was defined as the 95th percentile of the serum M-CSF level from the female control group (1277 pg/ml). Pretreatment serum M-CSF levels from the metastatic breast cancer patients ranged from 82.2 - 3,019.8 pg/ml, and were found elevated above the upper limit of normal in 15 of 204 patients (7.35 %). Patients with elevated pretreatment serum M- CSF did not have a significantly different objective response rate, clinical benefit rate, or time to progression to hormone therapy; but these patients did have significantly reduced overall survival (median survival 10.0 months) compared to patients with normal serum M-CSF levels (median survival 24.3 months)(p = 0.007). In multivariate analysis with serum HER-2/neu included as a covariate, elevated serum M-CSF level remained a significant independent variable for reduced survival (p= 0.032). Conclusions: Pretreatment serum M-CSF levels were elevated in 7 % of metastatic breast cancer patients compared to healthy female control subjects, and these patients had significantly reduced overall survival. Serum M-CSF deserves further study to determine its predictive and prognostic biomarker potential in breast cancer patients.

No significant financial relationships to disclose.
Association between expression of insulin resistance (IR) related genes and breast cancer outcome
Authors: Gennari A. , Sormani M. , Pronzato P. , Bruzzi P. , Ferrannini E. , Iozzo P. , Roncella M. , Ghilli M. , Mirisola V. , Pfeffer U.
Published: March 2020

10597

Background: IR is associated with adverse outcome in breast cancer. IR may affect patient outcome via the insulin and the IGF axis, acting through the tyrosine kinase signaling in enhancing cell proliferation. To date, no study explored the role of insulin- related gene expression in breast cancer progression. The aim of this study was to evaluate the prognostic value of a set of insulin-related genes using published microarray datasets. Methods: IR genes were sorted according to the “insulin sensitivity gene set” previously defined in peripheral tissues of healthy subjects screened for IR by the euglycaemic insulin clamp technique. One-hundred-forty- three genes were used to develop an outcome predictor on a training set of 102 primary breast tumors, randomly selected from 159 patients in the GEO database ( GSE1456 ). The outcome predictor was validated on the remaining 57 patients. Primary outcome measure was relapse free survival (RFS). Univariate non-parametric Mann-Whitney U test was used to identify genes differentially expressed. Expression of genes significantly correlated with relapse was combined in a linear score. Patients were classified as low or high risk with respect to the median score. Kaplan-Meier curves were used to evaluate the discriminating power of the score. Results: Fourteen genes resulted differentially expressed in the training set: the 8-year RFS was 91% (SE =4%) and 51% (SE = 8%) in the low risk and high risk group, respectively (p < .001). In the validation set, the 8-year RFS was 97% (SE = 3%) and 54% (SE = 10%), respectively (p = .009), supporting the discriminating ability of the score. By multivariate Cox analysis, the prognostic ability of the score was independent of subtype and grade (p < .001). Conclusions: Our data provide the first evidence that expression of insulin related genes can predict outcome, and indicate that the insulin pathway is involved in breast cancer progression. This is particularly important since IR is responsive to lifestyle interventions. We are currently validating the insulin-related score on independent breast cancer microarray datasets.

No significant financial relationships to disclose.
A new estrogen receptor on ER-negative breast cancer?
Authors: Peralta E. , Paris B. D. , Kamel O. W. , Louis S. , Dunnington G. L.
Published: March 2020

10604

Background: Premenopausal women and BRCA-1 mutation carriers have estrogen receptor-negative (ER-) breast cancers that paradoxically seem responsive to hormonal deprivation such as oophorectomy. GPR30 is a G protein-coupled receptor that is activated not only by estradiol but also tamoxifen and fulvestrant. The prevalence and significance of GPR30 in breast cancer are unknown. We hypothesized that GPR30 may be present in ER-negative tumors and may promote growth of ER-negative breast cancer in premenopausal patients. Methods: An immunohistochemical staining protocol for rabbit polyclonal GPR30 antibody (Novus Biological) was developed initially on the known GPR30-positive breast cancer cell line MCF-7 and the GPR30-negative breast cancer cell line MDA-MB-231 and later on a panel of human breast tumors from archived paraffin blocks. After an automated protocol was optimized, under an IRB-approved protocol all incoming breast cancer specimens were stained for GPR30 and reviewed by a pathologist blinded to the ER/PR status. Significance of associations was tested by chi square analysis. Results: Twenty-seven breast cancer tumors were studied. The mean age of the patients was 53 (range 20–87). All of 10 triple negative (ER-,PR-, HER2 neu-) and 2 ER-/Her2 neu+ tumors stained positive for GPR30. Only 4 of 11 ER+/Her2neu - tumors stained positive for GPR30 (p=0.004). The mean age of triple negative patients was 43.7 yr, while the mean age of ER+/Her2 neu- was 63.2 (p=0.02). Three out of 4 ER+/HER2 neu+ tumors stained positive for GPR30. Four patients were African Americans, 2 had triple negative tumors and 2 ER+/Her2+, all 4 were positive for GPR30. Conclusions: GPR30 appears to be frequently expressed in ER-negative breast cancer and may represent an adverse factor. ER+ tumors with good prognostic features (PR+, HER2 neu-) were mostly negative for GPR30, while high-risk tumors were positive for GRP30. Premenopausal status and African-American race are known risk factors for lower survival. In this study, the African-American patients had high-risk tumors that were also positive for GPR30. A longer- range study is needed to test the association of GPR30 expression with recurrence and death. Future studies to assess the function of GPR30 in breast cancer cells may yield prognostic and therapeutic applications.

No significant financial relationships to disclose.
In vitro abrogation of acquired tamoxifen resistance using histone deacetylase inhibitors
Authors: Beeram M. , Russell D. , DeGraffenreid L. , Freeman J.
Published: March 2020

10607

Tamoxifen (TAM) is a highly effective therapy for breast cancer. However, only 60% of estrogen receptor expressing (ER+) breast cancers respond to TAM (40% de-novo resistance). Continuous TAM treatment causes emergence of acquired resistance in these initially- responsive cancers. Strategies that abrogate TAM-resistance are therefore of significant clinical importance. One approach being studied is the use of histone deacetylase (HDAC) inhibitors. These compounds impact tumor biology both at the level of gene transcription and protein stability. In our study, we developed an in vitro model of tamoxifen resistance to test the efficacy of a hydroxamic acid derivative, Trichostatin A (TSA) at modulating breast cancer cell growth both in TAM responsive as well as TAM resistant cells. Forty-eight MCF-7 breast cancer single-cell colonies were isolated from a heterogeneous population, with 16 of the 48 (33%) surviving 4 months of continual growth in charcoal-stripped media with 10-7 M tamoxifen. ER expression was lost in 9 (56%) and HER-2 expression was acquired in 7 (44%) of the 16 resistant clones. The ER-negative cells demonstrated a concomitant increase in p-Rb, p21 and p27 levels, as well as significant increase in activation of both the PI3K and MAP kinase pathways. These results suggest that alteration of parent cell phenotype is an adaptive event at the transcriptional level, and is a significant mechanism for mediating TAM resistance. Inhibition of growth, by TSA, was studied in both the ER+ and ER- cells, with and without cotreatment with TAM. Cotreatment with TSA effectively blocked the emergence of resistance and prolonged the duration of response to TAM. The molecular mechanisms mediating this enhanced response were studied and focused on the effects of TSA on ER and HER-2 activity and expression. Our results indicate that short term exposure to TSA induces suppression of ER gene transcription, as well as targets ER for protein degradation where as long term exposure shows paradoxical effects at the genomic level. The effects of TSA on acquired HER-2 expression are being studied. These data have important clinical ramifications for the clinical development of HDAC inhibitors for the treatment of breast cancer patients.

No significant financial relationships to disclose.
The TLR4 polymorphism predicts the response of breast cancer patients to anthracyclins
Authors: Ghiringhelli F. , Apetoh L. , Tesniere A. , Andre F. , Lidereau R. , Delaloge S. , Chompret A. , Kroemer G. , Zitvogel L.
Published: March 2020

10612

Background: Conventional anticancer therapy mainly relies upon chemotherapy which supposedly directly eliminates tumor cells. However, we showed in mice that chemotherapy-induced tumor cell death markedly enhanced T-cell mediated tumor rejection, suggesting a role of innate immune receptors in the response to anthracyclins. Moreover, anthracyclins are less effective in athymic nude or Toll like receptor-4 (TLR4) deficient mice (Obeid et al. Nat. Med. 2006). A single nucleotide polymorphism (SNP) in TLR4 (896A/G, Asp299Gly) affecting the extracellular domain of TLR4 is associated with reduced endotoxin responses in humans. We addressed whether the TLR4 SNP could affect the response to anthracyclins in breast cancer patients. Methods: We included 280 patients from three cancer centers bearing non-metastatic breast cancer presenting with lymph node involvement and treated with anthracyclin-based regimen after local surgery. TLR4 genotyping performed on leukocytes (PCR based on exonuclease degradation of dual labelled allele-specific oligos) and statistical analyses were performed in a blinded fashion. Results: The frequency of Asp299Gly (heterozygote) and Gly299Gly (homozygote) TLR4 germline polymorphisms was 17.1 and 0.7 % respectively. Patients that exhibited a mutated TLR4 allele did not differ from patients displaying the normal TLR4 allele for all classical prognostic factors (age, pathological tumor size, lymph node involvement, tumor grade, hormone receptors, and median follow up, p>0.05). However, the frequency of metastases was statistically higher in the mutated TLR4 group five years after surgery (40% versus 26.5% in non mutated patients, p <0.05, RR: 1.7, 95% CI [1.2–3.6]). Moreover, the Kaplan Meier estimate of metastasis-free survival indicated an overall significantly lower percentage of metastases-free patients in the mutated TLR4 group (Log rank test, p = 0.03). Conclusions: It is the first demonstration of the role of innate immunity and TLR4 in the response to anthracyclins in localized breast cancer.

No significant financial relationships to disclose.
A genomic signature of chemotherapeutic multidrug resistance provides a rational approach to identify patients for targeted therapy in advanced solid tumors
Authors: Anguiano A. , Mori S. , Chang J. , Vlahovic V. , Kelley M. J. , Ginsburg G. , Dressman H. , Nevins J. R. , Potti A.
Published: March 2020

10613

Background: Multidrug resistance (MDR), the process by which cells resist many structurally and functionally unrelated drugs, remains a major challenge for cancer treatment. Strategies to reliably predict MDR will revolutionize cancer therapy. Methods: We made use of in vitro drug sensitivity data on 47 FDA approved chemotherapeutic agents from the NCI-60 database coupled with Affymetrix microarray data, to develop a gene expression signature of chemotherapeutic MDR, using a 'metagene’ approach to gene expression analysis (Potti et al, Nature Medicine, 2006). The capacity of the MDR signature to predict in vitro and in vivo resistance was evaluated in multiple independent cell line and patient cohorts. Results: A gene expression signature of chemotherapeutic MDR developed from cancer cell lines using a novel ‘metagene’ approach can accurately predict patients resistant to standard chemotherapeutic agents, including multi-drug regimens. This includes cohorts of patients with early stage breast cancer (n = 171; accuracy: 75.5%, sensitivity: 49.3%, specificity: 92%), patients who received neoadjuvant chemotherapy (n = 51; accuracy: 78.5%, sensitivity: 73.7%, specificity: 92.3%), and ovarian cancer (n = 105; accuracy: 83.8%, sensitivity: 71.8%, specificity: 93.7%). We further show that MDR signature expression is an independent prognostic factor in early stage breast cancer (p = 0.02, odds ratio: 3.49) and a large cohort of non-small cell lung cancer (p = 0.03, odds ratio: 5.70), in a multivariate analysis. Finally, to identify a rational therapeutic strategy in patients with the MDR phenotype, we have integrated the prediction of MDR with profiles of oncogenic pathway deregulation (Bild A, Nature, 2006) and show that targeting the Src pathway using a Src specific inhibitor (SU6656) circumvents MDR in the in vitro setting. Conclusions: The development of a gene expression signature of MDR, that can predict resistance to commonly used cytotoxic chemotherapies, provides a rational approach to developmental therapeutics, by identifying novel 'customized’ candidate drugs (e.g. targeting Src) for further clinical development.

No significant financial relationships to disclose.
Phase I/II trial combining the HDAC inhibitor, valproic acid (VPA) and FEC100 (5-fluorouracil, epirubicin and cyclophosphamide) in locally advanced/metastatic breast cancer
Authors: Munster P. N. , Marchion D. C. , Schmitt M. , Bicaku E. , Lacevic M. , Minton S. , Carter W. , Daud A.
Published: March 2020

1065

Background: Cell culture and xenograft studies suggest a synergistic interaction between histone deacetylase inhibitors (HDACi) and topoisomerase (topo) inhibitors, as well as other DNA targeting agents. Methods: In this phase I/II study, we determined the effects of escalating doses of VPA on the clinical efficacy and tolerability of epirubicin. The phase I part was open to patients will all solid tumors. A limited phase II part at the maximum tolerated dose (MTD) of VPA enrolled 10 breast cancer patients and incorporated the breast cancer regimen FEC100 (5-fluorouracil, epirubicin, cyclophosphamide (600/100/600 mg/m2)). VPA was given on days 1–3 prior to epirubicin/FEC100 in 3-week cycles. HDAC expression, histone acetylation and topo II expression were evaluated in pre-and post-VPA peripheral blood mononuclear cells and tumor samples. Results: Fifty-four (44 in phase I and 10 in phase II) patients [median age 55 (39–78)] received VPA (mg/kg/day): 15, 30, 45, 60, 75, 90, 100, 120, 140 and 160. Tumor types included: breast (10+10), melanoma (11), lung (6), sarcoma (2), GYN (2), GI (5) and others (8). Dose-limiting toxicities included somnolence, confusion and febrile neutropenia. No exacerbation of FEC100/epirubicin-related toxicities was observed. Objective responses in the phase I part 9/41 (22%) were seen across different tumor types despite a median number of 3 (0–6) prior regimens with stable disease/minor response in 16/41 (39%). In the breast-specific phase II part, partial responses to date were seen in 4/8 (50%) and stable disease in 2/8 (25%), progression in 1/8 (12.5%), 1/8 (12.5%) patients withdrew consent. All breast cancer patients with a response/stable disease received the maximal number of seven cycles. VPA plasma concentrations correlated with VPA dose. There was a positive correlation between histone acetylation and VPA dose as well as plasma levels in PBMC and further correlated with those in tumors. Conclusion: A sequence-specific combination of VPA and FEC100 in breast cancer is highly active without exacerbation of chemotherapy-induced toxicities. A neoadjuvant phase II trial using VPA (120 mg/kg) -> FEC100 in patients with early stage breast cancer is ongoing.

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Retrospective analysis of data from a phase I trial with a new adaptive method
Authors: Meille C. , Favier L. , Coudert B. , Barbolosi D. , Iliadis A. , Biville F. , Garnier J. , Ferri-Dessens R.
Published: March 2020

1067

Background: The phase I trial CAPEV CGFL 01 [ESMO, Vienne, Austria: 2004.V 52–53] aimed to find the Maximum Tolerated Dose (MTD) of i.v. Vinorelbine when associated with oral Capecitabine for three cycles of 21 days in metastatic breast cancer (MBC). Method: Vinorelbine was administered at d1 and d15 and Capecitabine b.i.d. from d1 to d14 (fixed at 2,000 mg/m2/day). Trial design was based on “3+3” scheme with predetermined dose levels of Vinorelbine starting at 25 mg/m2/day. Ten patients (age 46–67 years) with MBC were enrolled in this trial. A new phase I adaptive method [Statistical Methods in Biopharmacy. Statistical Innovations in Clinical trials, Paris September 2005] under validation was used to analyze data available from the CAPEV trial. This method performs precise definition of Dose Limiting Toxicity (DLT) and modeling of all the grades of toxicity. The method allows sequential real time analysis of data by alternating estimation and design steps. Results: Most frequent toxicities were hematological (anemia and neutropenia) and cytolysis. Three DLTs occurred for the first 6 patients included at the starting level of Vinorelbine. De-escalation to 20 mg/m2 was done at the 7th patient but a new DLT occurred for the 7th patient and lead to trial ending at the 10th patient. Firstly, by analyzing all available data, Vinorelbine MTD was calculated at 20.2±4.04 mg/m2/day. Secondly, data was processed according to the inclusion time of patients; the adaptive method calculated a Vinorelbine MTD equal to 19.5±8.34 mg/m2/day as early as the 4th patient. Conclusion: The administration scheme of i.v. Vinorelbine and oral Capecitabine tried in this study cannot be retained for MBC treatment. However, early convergence of adaptive method clearly shows its high performance in the de-escalation scheme and Vinorelbine MTD determination. With this method Vinorelbine MTD could have been determined with the 4th patient data without needing the inclusion of 6 additional patients (4th and not 7th patient). This method should be used in prospective phase I trials and in parallel with classical and clinical method of MTD determination.

No significant financial relationships to disclose.
Bortezomib and capecitabine in patients with metastatic breast cancer previously treated with taxanes and/or anthracyclines: Final results of a phase I/II study
Authors: Schmid P. , Regierer A. , Kiewe P. , Schippinger W. , Greil R. , Lehenbauer-Dehm S. , Niederle N. , Freier W. , Van de Velde H. , Possinger K. , Kuehnhardt D.
Published: March 2020

1072

Background: Capecitabine has shown substantial activity in taxane and/or anthracycline pretreated breast cancer patients. Bortezomib, a 26S proteasome inhibitor, has been shown to increase sensitivity to chemotherapy. This phase I/II trial was initiated to evaluate the combination of capecitabine and bortezomib in heavily pretreated patients with metastatic breast cancer. Methods: Patients with metastatic breast cancer and prior taxane and/or anthracycline therapy were treated with bortezomib (1.0–1.3 mg/m2; days 1, 4, 8 & 11) and capecitabine (1,500–2,500 mg/m2, days 1–14) in 3-weeks intervals for up to 8 cycles. Primary endpoints were to determine the optimal doses for the combination (phase I) and the tumor response rate (RR) (phase II). Secondary endpoints included safety, time to progression (TTP), duration of response (DR), and overall survival (OS). Results: A total of 35 patients were enrolled and 29 patients were assessable for response. The majority of patients had received 2 or 3 lines of chemotherapy (69% and 14%, respectively) prior to the study. The maximum tolerated doses (MTD) were bortezomib 1.3 mg/m2 and capecitabine 2500 mg/m2. Dose limiting toxicities were Grade 3 stomatitis in 1 out of 6 patients at 1.0/2,000 and Grade 3 diarrhea in 1 out of 6 patients at 1.3/2,500. Myelosuppression was low. Non-hematological toxicities were generally mild to moderate with no G4 toxicity being observed. Most common side effects were thrombocytopenia (Grade 3/4 27% of patients), diarrhea (18%), hand-foot syndrome (12%), peripheral neuropathy (12%), leucopenia (9%) and asthenia (9%). The overall RR was 17.2% and an additional 31% of patients had stable disease (31%; 4 unconfirmed). In the 21 patients treated at the MTD, RR was 17.6% and 47% of patients had SD. Median TTP and OS were 3.5 months (95% CI 1.9–4.4) and 7.5 months (95% CI 5.6–14.6), respectively. Median DR was 4.4 months. Conclusions: The combination of bortezomib and capecitabine is well tolerated and has moderate antitumour activity in heavily pretreated breast cancer.

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Fulvestrant in advanced breast cancer following following failure of tamoxifen and a third generation aromatase inhibitor
Authors: Wang J. , Jain S. , Heller W. , Mackie D. , Watson V. , Dweck M. , Coombes R. C. , Palmieri C.
Published: March 2020

1073

Background: Endocrine therapy is a key modality in the management of estrogen receptor positive metastatic breast cancer. Fulvestrant (ICI 182,780) is an estrogen receptor downregulator. It has previously been shown to be as effective as anastrozole in patients who had previously progressed on tamoxifen. Methods: A retrospective study was carried out of metastatic breast cancer patients treated at Charing Cross Hospital between 2002–2005 who had received fulvestrant following treatment failure with tamoxifen and a third generation aromatase inhibitor. All patients were postmenopausal and received fulvestrant 250mg IM every 28 days. Measurable disease was assessed by response evaluation criteria in solid tumors (RECIST). Results: A total of 45 patients were identified with a median age of 60 (range 36 to 90). The ER status was known in 95% (n=43) of patients and was positive in all cases, it was unknown in 2% (n=2). At the time of commencing fulvestrant, 96% (n=43) had metastatic disease and 4% (n=2) locally advanced disease. All patients had received at least 2 lines of prior endocrine therapy (including adjuvant therapy), at time of starting fulvestrant the median number of prior regimens was 3 (range 3–5). Fulvestrant was administered for a median of 4 months (range 1 to 20 months), with 4 patients currently still receiving therapy as of 1 November 2006. Of the 45 patients, 2.2% (n=1) achieved a partial response, while 31% (n=14) achieved stable disease for at least 6 months. Thus, 33.3% (n=15) obtained clinical benefit (defined as PR or SD for at least 6 months). The response rates based on line of therapy will be presented. Of the 45 patients, 41 were evaluable for survival data. The median survival of the remaining patients from the start of fulvestrant therapy was 9 months (range 1 to 48 months). Of the 44 patients, 14% (n=6) remain alive. The treatment was well tolerated and toxicity data will be presented. Conclusions: Fulvestrant is well tolerated and is efficacious as treatment for advanced breast cancer that has failed tamoxifen and a third generation aromatase inhibitors.

No significant financial relationships to disclose.