bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of FOXC1 in regulation of basal-like/triple-negative breast cancer
Authors: Ray P. S. , Wang J. , Qu Y. , Shin-Sim M. , Shamonki J. , Liu B. , Hoon D. S. , Giuliano A. E. , Cui X.
Published: August 2020

11016

Background: Class identification studies have proposed 3 prognostically relevant molecular subtypes of breast cancer: luminal, HER2 and basal-like. The latter is associated with poor prognosis but its molecular basis is not clear. We hypothesized a direct correlation between FOXC1 expression and basal-like breast cancer. Methods: Expression of FOXC1, CK5, CK14, EGFR, c-Kit, αB-crystallin, ITGB4 and FOXC2 in basal-like breast cancer was examined using publicly available microarray datasets. A molecular signature of 40 genes sharing co-ordinate up or down regulation with FOXC1 was identified on one microarray (49 patients) and validated on 5 other microarrays (1,232 patients). The clinical significance of FOXC1 gene expression and the FOXC1 gene signature was evaluated using censored survival data. FOXC1 protein expression was assessed by immunohistochemistry (IHC) of a 96-sample breast cancer tissue microarray. Normal breast epithelial, luminal and basal breast cancer cells transfected with FOXC1 vectors were evaluated for cell proliferation, migration and invasion. Results: FOXC1 was found to be consistently and exclusively upregulated in basal-like triple negative breast cancer and was associated with poor overall survival (p<0.0001). The FOXC1 gene signature accurately predicted the basal-like phenotype. IHC analysis of FOXC1 protein expression in human breast cancers confirmed its potential to be used as a clinical biomarker of basal-like breast cancer. Normal breast epithelial cells and luminal breast cancer cells with low or no FOXC1 expression underwent epithelial-to-mesenchymal transition and displayed increased cellular proliferation, migration, invasion, and expression of basal cell markers when FOXC1 was overexpressed. In contrast, knockdown of FOXC1 by shRNA in basal-like breast cancer cells conferred luminal phenotype. Breast cancer progression-linked signaling pathways like NF-κB and p38MAPK were significantly stimulated in basal-like breast cancer as well as by in vitro FOXC1 overexpression. Conclusions: FOXC1 is a dominant determinant of the basal-like phenotype of breast cancer. We propose FOXC1 to be the single best molecular marker of and a potential therapeutic target for basal-like / triple negative breast cancer.

No significant financial relationships to disclose.
CYFRA 21–1 (CYFRA) as a prognostic and predictive marker in advanced non-small cell lung cancer (NSCLC): CALGB 150304
Authors: Rosenblatt P. Y. , Edelman M. J. , Christenson R. H. , Hodgson L. , Wang X. , Kratzke R. , Vokes E.
Published: August 2020

11020

Background: Cytokeratin 19 and its soluble fragment CYFRA have been studied as markers that may correlate with response to therapy and survival in NSCLC. As part of CALGB 30203, a randomized trial of carboplatin/gemcitabine with eicosanoid modulators (celecoxib, zileuton or both) in advanced NSCLC (Edelman JCO 2008), serum CYFRA levels were obtained prior to and during treatment. The objective of this study was to evaluate the possible correlation of CYFRA as a predictive and prognostic marker and to confirm a previous finding that a 27% reduction in CYFRA after one cycle (21 days) of treatment correlated with a longer survival (Vollmer Clin Ca Res, 2003). Methods: Paired specimens were available from 88 patients. CYFRA was measured at baseline and after cycles 1 and 2 using an electrochemoluminescent assay (Roche Diagnostics) on the ElecSys 2010 system as previously described. Using logarithm of the initial concentration and logarithm of the difference in concentrations, we analyzed these in relation to overall survival (OS) and failure free survival (FFS). Results: 1. Lower baseline CYFRA levels were associated with both longer overall survival and failure free survival (p = <0.0001 and p=0.0045). 2. Larger reductions in CYFRA levels correlated to longer overall survival and failure-free survival (p=0.0254 and p=0.0298). 3.We failed to replicate that a drop of >27% in CYFRA levels had statistical significance in overall or failure free survival (p=0.6489 and p=0.9636). Conclusions: CYFRA and change in CYFRA appear to be reliable markers in predicting the response to chemotherapy for nonsmall cell lung cancer; however, a precise threshold to mark response has yet to be determined.

No significant financial relationships to disclose.
Clinicopathologic features of EML4-ALK mutant lung cancer
Authors: Shaw A. T. , Costa D. , Mino-Kenudson M. , Digumarthy S. , Yeap B. Y. , Admane S. , Rodig S. , Chirieac L. , Iafrate A. J. , Lynch T. J.
Published: August 2020

11021

Background: EML4-ALK is a novel fusion oncogene in non-small cell lung cancer (NSCLC). The fusion results from a small inversion within chromosome 2p, leading to expression of a constitutively activated, chimeric tyrosine kinase. The clinicopathologic features of these patients have not been definitively established. Furthermore, the clinical outcome of these patients is unknown. Here we present the largest series of EML4-ALK mutants to date, and the first analysis of treatment response and survival in metastatic patients with and without EML4-ALK. Methods: Patients with NSCLC were selected for genetic screening based on 2 or more of the following characteristics: female gender, Asian ethnicity, never or light smoking history, and adenocarcinoma histology. The EML4-ALK inversion was identified using FISH and confirmed by IHC for ALK expression. EGFR mutation status was determined by direct sequencing of EGFR exons 18–21. Results: Of 141 patients screened, 18 (13%) were ALK mutant, 31 (22%) were EGFR mutant, and 92 (65%) were wild-type (WT) for both ALK and EGFR. The majority of tumors were adenocarcinomas, with ALK but not EGFR mutant tumors strongly associated with the signet ring cell subtype. Compared to the EGFR mutant and WT cohorts, patients with ALK mutant tumors were significantly younger (median age 52.5 vs 64 yrs, p=0.003), and more likely to be male (61% vs 30%, p=0.015). ALK mutants, like EGFR mutants, were also more likely to be light/never smokers compared with WT patients (100% vs 43%, p<0.001). Among patients with metastatic disease, there was a significant association between EML4-ALK and resistance to EGFR TKIs, with no responses by RECIST in the ALK cohort. ALK mutants and WT patients showed similar response rates to platinum-based chemotherapy (60% vs 65%, p=1), and had similar 1-yr survival rates (81% vs. 66%, p=0.43). Conclusions: EML4-ALK defines a new molecular subset of NSCLC with distinct clinical and pathologic characteristics. The frequency of EML4-ALK is particularly high in light/never smokers without EGFR mutation. These patients do not benefit from EGFR TKIs and should be treated with other standard agents or ALK targeted therapies.

[Table: see text]
Combination effects of gefitinib plus cisplatin in non-small cell lung cancer (NSCLC): Why have phase III trials failed?
Authors: Tsai C. , Chen T. , Chang K. , Hsiao S.
Published: August 2020

11022

Background: Four phase III large scale randomized controlled trials combining standard chemotherapeutics and epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced NSCLC have failed to show benefit. The mechanism for the failure is still yet not clear. Methods: Seventeen NSCLC cell lines were tested; two had induced P-glycoprotein (Pgp) over-expression. Three of the 4 EGFR mutants exhibited activating mutations. All lines were simultaneously treated with gefitinib plus cisplatin (GC). Selected 4 lines were tested with paclitaxel plus cisplatin (PC) or gefitinib (PG) and three-drug combination (PCG). The MTT assay with application of classical isobole method and statistical analysis was used and dose-log response curves (DRCs) were examined for evaluating the possible resistance mechanisms. Results: We found that combined GC regimen had significant antagonism at the IC50 level (13/15 lines; mean CI=1.184±0.037, P=0.001). The mean CI values of PCG were higher than or comparable with those of PC or PG in either EGFR wild type or mutant cells. DRC analysis mainly showed non-saturable passive resistance, suggesting that gefitinib 0.001–0.3 μM could interfere with cisplatin cell entry in a dose dependent fashion when cisplatin concentrations over 1–3μM, thus leaded to antagonism. Giving gefitinib 24 hours after administration of cisplatin could abolish the antagonism. Conclusions: In NSCLC cells, combination of GC showed antagonistic interaction likely because gefitinib interfered with cisplatin cell entry. Three-drug combination PCG was not better than two-drug combination PC or PG in either EGFR wild type or mutant cells. Clinically, simultaneously combined EGFR TKI with platinum in NSCLC should be avoided regardless of EGFR mutation status.

[Table: see text]
Effects of G-CSF on circulating tumor cells (CTC) and CA 27.29 in breast cancer patients
Authors: Hepp P. , Rack B. , Schneider A. , Rezai M. , Tesch H. , Beck T. , Söling U. , Lichtenegger W. , Beckmann M. W. , Janni W.
Published: August 2020

11027

Background: Some recent publications indicated that the use of G-CSF could be connected to an increase in CTC as well as elevated levels of tumor markers such as CA 27.29. In the SUCCESS Trial CTC and CA27.29 are examined before and after adjuvant chemotherapy (CHT) in 3754 breast cancer patients (pts). Methods: The SUCCESS Trial is a phase III trial comparing FEC-Docetaxel vs. FEC-Doc-Gemcitabine regime and 2 vs. 5 years of treatment with zoledronate in patients with primary breast cancer (BC) (N+ or high risk). Blood samples are taken before and after CHT. CTC were assessed with the CellSearchSystem (Veridex, Warren, USA). After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labeled with anti-cytokeratin (8,18,19) and anti-CD45 antibodies to distinguish epithelial cells and leukocytes. CA27.29 has been measured with ST AIA-PACK Ca27.29 reagent using MUC-1 for AIA-600II (Tosoh Bioscience, Tessenderlo, Belgium). The cutoff for CA27.29 is 32 U/ml and >1 cell for the CTC analysis. Patients were grouped to CTC/CA27.29 raise or no raise and 1 to 6 cycles with G-CSF or no G-CSF at all. Results: Data on 1510 pts are available for CTC analysis. 745 pts (49%) received at least one course of G-CSF. 117 pts (8%) showed an increase in CTC after CHT. In this group 52 (3%) pts received G-CSF and 65 (4%) did not. 693 pts with stable or decreased CTC received G-CSF (46%) and 700 did not (46%). There was no significant difference (p=0.29). The analysis of CA27.29 is based on the data of 2556 pts. 1252 pts (49%) received at least one course of G-CSF. 338 pts (13%) exceeded the threshold for CA27.29 only after CHT. In this group 209 pts (8%) received G-CSF and 129 (5%) did not. 1043 pts with stable or decreased CA27.29 received G-CSF (41%) and 1175 did not (46%). This difference was highly significant (p<0.0001). Conclusions: No evidence can be provided for a significant correlation between an increase in the number of CTC and the application of G-CSF over CHT. Nevertheless the results on CA27.29 showed a highly significant correlation between the administration of G-CSF and elevated CA27.29 levels directly after CHT. This could be a possible explanation for the often observed increase of tumor markers after CHT.

[Table: see text]
Preclinical testing of the PARP inhibitor ABT-888 in microsatellite instable colorectal cancer
Authors: Vilar Sanchez E. , Chow A. , Raskin L. , Iniesta M. D. , Mukherjee B. , Gruber S. B.
Published: August 2020

11028

Background: Microsatellite instability (MSI) represents approximately 15% of colorectal cancer (CRC) cases. MSI due to hypermethylation or mutation in DNA mismatch repair genes leads to genetic instability and a mutator phenotype. Genetic instability is particularly high at repetitive sequences such as those located in MRE11, RAD50, CtIP and MBC. Each of these genes are implicated in the double strand break (DSB) repair pathway. PARP inhibitors induce single strand breaks that remain unrepaired and then will be converted to DSB during DNA replication. Our objective was to assess the preclinical activity of a novel PARP inhibitor ABT-888 in MSI cell lines deficient in the DSB repairing pathway and compare it to Microsatellite Stable (MSS) lines. Methods: We used the systems biology tool “Connectivity Map” to synthesize data from 5 different published studies of expression profiling of MSI CRC phenotype and to identify target compounds. We assessed the mutational status of MRE11, RAD50, CtIP and MBC in a panel of 10 CRC cell lines displaying either MSI or MSS, and measured the expression of MRE11 by quantitative RT-PCR. We tested the cytotoxic activity of single-agent ABT-888 for 6 days in MSS and MSI cell lines, stratified by mutational status. Flow cytometry was performed after 24 hours. Results: Systems biology studies identified PARP inhibitors as a candidate compound relevant for MSI CRC. Mutational status of MRE11 was perfectly correlated with MSI status. ABT-888 shows a preferential activity on those MSI cell lines harboring mutations in both MRE11 and RAD50 genes compared to MSS cell lines (wild-type for both genes). A significant correlation exists between MRE11 expression levels and cytotoxicity to ABT-888 at 10 μM (R2=0.915, P<0.001). Flow cytometry analyses show a G1 arrest following to the treatment with ABT-888 that is higher in MSI cell lines with mutations in MRE11 and RAD50 compared to MSS cell lines. Conclusions: This is the first report of the preclinical activity of a PARP inhibitor in CRC models. MSI colorectal tumors deficient in DSB repair show a higher sensitivity to PARP inhibition. Further clinical investigation of ABT-888 as a single agent or in combination with other chemotherapy drugs inducing DSB is warranted in MSI CRC with mutations in MRE11 and RAD50.

No significant financial relationships to disclose.
Detection of BRAF kinase mutations in melanoma, ovarian, and prostate carcinomas: Evidence for tumor heterogeneity in clinical samples
Authors: Litterman A. J. , Yancovitz M. , Shapiro R. , Berman R. , Pavlick A. , Daarvishian F. , Blank S. , Lee P. , Osman I. , Polsky D.
Published: August 2020

11031

Background: Several studies have provided evidence that solid tumors are polyclonal malignancies, an observation which may contribute to difficulties in achieving durable treatment responses. In some patients, molecularly targeted therapies may be compromised due to heterogeneity among tumor subclones. In this study we compared conventional DNA sequencing with a fluorescent-based mutant-specific PCR (MS-PCR) assay to detect the BRAF hotspot mutation V600E in a large panel of patient tumors, including paired primary and metastatic tumors from individual patients. Methods: BRAF MS-PCR and conventional sequencing were performed on DNA from 304 tumors (112 melanoma, 110 ovarian, 82 prostate) to determine the presence of the BRAFV600E hot-spot mutation. Among the melanomas were 18 matched primary and metastatic specimens, and 40 metastatic specimens from 19 patients, each of whom had 2 or more metastases. Results: DNA sequencing detected mutations in 5/110 (4.5%) ovarian tumors, 1/82 (1.2%) prostate tumors, and 36/112 (32%) melanomas. In contrast, the MS-PCR assay detected mutations in 12/110 (11%) ovarian tumors, 15/82 (18%) prostate tumors and 85/112 (76%) melanomas. The presence of contaminating normal tissue was scored for each melanoma sample, but excess normal tissue did not influence the results using either methodology. In all cases mutations detected by sequencing were also detected by MSPCR. Among 18 patients with matched primary and metastatic melanoma, 8/18 (44%) had discordant results including 2 patients with mutant primary tumors and wild-type metastases; among the 19 patients with multiple metastases 5/19 (26%) had discordant (both wild-type and mutant) tumors. Conclusions: Using a highly sensitive BRAF mutation detection method, we observed substantial evidence for heterogeneity within clinical tumor specimens. This was especially true in melanoma samples, where multiple specimens from individual patients differed with respect to the presence of the mutant BRAF allele. These results suggest that failures of molecularly targeted therapies, such as those directed against mutant BRAF, may be due in part to a lack of clonality among the tumors under treatment.

No significant financial relationships to disclose.
Quantitative determination of predictive cancer biomarkers in formalin-fixed, paraffin-embedded tissue using a new, fully automated method for RNA isolation
Authors: Müller B. M. , Kronenwett R. , Hennig G. , Weber K. , Bohmann K. , Winzer K. J. , Kristiansen G. , Petry C. , Dietel M. , Denkert C.
Published: August 2020

11032

Background: Personalized cancer therapy depends on the evaluation of tissue-based biomarkers in routine tumor samples. As many new biomarkers are measured on the mRNA level, standardized procedures for mRNA analysis in formalin-fixed, paraffin-embedded (FFPE) tissue are needed. In this study, we present a novel, standardized, and fully automated method for fast isolation of total RNA from FFPE tissue sections. Methods: Tissue samples (n = 501) from 167 breast carcinomas which had been stored between 2 months and 21 years were investigated. Total RNA was extracted from tissue sections using a new method based on silica-coated iron oxide beads in combination with a specific liquid-handling robot. Novel and unique features of the method are an automated extraction-integrated deparaffinization step and a bead-based negative selection step to eliminate any undigested tissue. Yield of RNA was assessed using the Ribogreen assay. mRNA fragment lengths were estimated by reverse transcription PCR (RT-PCR) for G6PDH. Expression of the breast cancer biomarkers ESR1, PGR and HER2 was measured by kinetic RT-PCR (kPCR) and compared with immunohistochemistry (IHC). Results: RNA was successfully isolated from all samples, with a mean yield of 1.4 μg/sample (range: 0.1–7.8 μg) and fragment lengths of at least 150 bp in 99% of samples. PCR analyses could be performed in all samples. Investigating three sections of each tumor, we observed a low section-to-section variability of kPCR results (root of mean squared errors of relative ESR1, PGR or HER2 expression in three sections: 0.2–0.5 Ct values). Comparing kPCR results with IHC, we detected a good concordance between both methods, with agreements of 98.4% (standard error [SE]: 2.7%) for ESR1, 84.4% (SE: 5.3%) for PGR and 89.9% (SE: 8.9%) for HER2. Conclusions: This novel RNA extraction method is a major technical improvement for implementation of reproducible, high-throughput and cost-efficient testing of cancer biomarkers in the clinical routine and in gene-expression research studies using archived FFPE material in molecular labs.

[Table: see text]
Thymidilate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and dihydrofolate reductase (DHFR) as predictive markers of capecitabine efficacy in breast cancer patients
Authors: Audet R. , Shen C. , Edgerton S. , Lay L. , Vang Nielsen K. , Thor A. D. , Chang J. C. , Miller K. D. , Sledge G. W. , Leyland-Jones B.
Published: August 2020

11033

Background: The efficacy of 5-FU and the pro-drug capecitabine (C) is mediated through the thymidylate synthase pathway and may be affected by expression of key enzymes in this pathway. The expression and/or gene copy number of TS, TP, DPD and DHFR was assessed and correlated with time-to progression (TTP) and progression-free survival (PFS). Methods: Adult female patients with pathologically confirmed breast cancer and locally advanced or metastatic disease were treated with C 1000 mg/m2 BID days 1–14 of a 21-day cycle. Formalin-fixed, paraffin-embedded (FFPE) specimens from C-treated patients were processed for RNA extraction. Total RNA was isolated from 32 FFPE tissue samples containing at least 70% tumor cells and RNA levels for TS, TP, DPD and DHFR were quantified using real time RT-PCR and Affymetrix GeneChip microarrays. Custom made TS and TP FISH probes (Dako, Glostrup, Denmark) were used to evaluate gene copy number and gene to reference ratios in at least 60 morphologically intact non-overlapping nuclei. Markers were correlated with TTP and PFS using Cox proportional hazard models in 24 patients. Results: Higher TS gene copy number was significantly associated with a decrease in PFS (HR 1.46, 95% CI 1.08 to 1.96, p=0.014) and TTP (HR 1.49, 95% CI 1.05 to 2.13, p=0.028). However, the association between TS RNA levels (expressed as Ct values) and PFS (HR 0.82, 95% CI 0.61 to 1.11, p=0.198) or TTP (HR 0.74, 95% CI 0.49 to 1.14, p=0.172) failed to reach statistical significance. RNA levels, determined by Affymetrix, were significantly correlated with RT-PCR for TS (r= -0.5073, p=0.0004) and DHFR (r=-0.50994, p=0.003). Neither TP gene copy number nor RNA levels were significantly associated with PFS (HR 099, and HR 0.93) or TTP (HR 1.07 and HR 1.029), respectively. The association between RNA levels and PFS or TTP did not reach statistical significance for both DPD (HR 1.17 and HR 1.13) and DHFR (HR 0.68 and HR 0.63), respectively. Conclusions: These data indicate that TS gene copy number, assessed by FISH with proper standardization, might be a useful and easily accessible marker for C sensitivity in human breast cancer and warrants further investigation.

[Table: see text]
Cell-free miR-10b and miR-21 as diagnostic biomarkers for malignant effusions
Authors: Xie L. , Chen X. , Qian X. , Wei J. , Ding Y. , Zhang C. , Liu B.
Published: August 2020

11035

Background: Numerous studies have proved the roles of tissue microRNAs (miRNAs) as biomarkers in cancer diagnosis. Cell-free miRNAs from plasma or serum have been shown to be good biomarkers in the diagnosis of cancers. Here we tried to explore the potential application of cell-free miRNAs as diagnostic biomarkers to discriminate malignant effusions from benign. Methods: To obtain a molecular signature for malignant effusions, a stem-loop real-time PCR was adopted to measure the expression of 23 tumor-related miRNAs. After a pre-screening for discarding low amount miRNAs in 10 samples (5 benign/5 malignant) and a proper internal control was identified, the expression levels of 7 miRNAs were further compared among 74 malignant effusion samples (35 gastric cancer-related ascites and 39 lung cancer-related plural effusions) and 30 benign effusions. miRNAs specifically associated with malignant effusions were derived from a training group (20 malignant/10 benign) and tested in a validation group (54 malignant/20 benign). All samples were confirmed by both pathological and clinical diagnosis. Results: miR-215 was employed as the reference RNA for its abundance and consistence. The expression of miR-10b was significantly different between benign and malignant effusions (P< 0.001). Furthermore, it was showed that miR-10b can distinguish malignant plural effusions with high accuracy (areas under the curve (AUC) of receiver operating characteristic (ROC) curve 0.98, 95% CI, 0.90 to 1.0, P< 0.001), and miR-21 can be used to discriminate malignant ascites (AUC of ROC curve 0.71, 95% CI, 0.56 to 0.83, P= 0.018). Conclusions: In the present study, we demonstrated that cell-free miRNA can serve as a novel diagnostic biomarker in distinguishing malignant from benign effusions.

No significant financial relationships to disclose.
Concordant HER2 status between malignant CSF cells and primary breast carcinoma tissue
Authors: Kwon Y. , Ro J. , Lee K. S. , Park I. H.
Published: August 2020

1104

Background: Leptomeningeal diseases (LMD) from breast cancer are detected in up to 19% of patients with brain metastasis and their prognosis is extremely poor. Diagnosis of leptomeningeal disease requires positive cerebrospinal fluid (CSF) cytology. However, it is not known whether HER2 status of malignant CSF cells coincides with that of original breast carcinoma cells. We intended to determine whether CSF cytology specimen is suitable for evaluating HER2 status by fluorescence in situ hybridization (FISH) in LMD to provide the basis for HER2 targeted intrathecal therapy. Methods: Both formalin-fixed paraffin-embedded (FFPE) breast carcinoma tissue and liquid based CSF cytology specimen were tested for HER2 status in 16 patients who developed LMD at National Cancer Center between Dec 2004 and Jul 2008. We evaluated HER2 gene amplification by FISH on destained CSF cytology slides which contained minimum 20 malignant cells per slide, and compared with HER2 status by immunohistochemistry (IHC) in FFPE breast carcinoma tissue. HER2 was determined positive when FISH ratio > 2.2 or IHC 3+ according to ASCO/CAP guideline. Results: Concordance rate of HER2 status between CSF cytology by FISH and FFPE tissue by IHC was 100% as shown in the table. Conclusions: When CSF cytology specimen was appropriately prepared yielding adequate cellularity without dry artifact, CSF cytology was suitable for evaluating HER2 status by FISH in LMD. Intrathecal HER2 targeted therapy could be attempted when FFPE breast carcinoma tissue is HER2 positive in view of highly concordant HER2 status by our data. Supported by NCC Grant No 0610240- 3.

[Table: see text]

No significant financial relationships to disclose.
Influence of loco-regional radiation therapy on subsequent cancer risk among BC pts with p53 germline mutations
Authors: Rahal A. , Caron O. , Bourgier C. , Frebourg T. , Bressac de Paillerets B. , Barreau L. , Pachet C. , Mathieu M. , Marsiglia H. , Delaloge S.
Published: August 2020

11043

Background: The management of breast cancer (BC) patients (pts) with germline p53 mutations, a rare genetic condition, is not the object of specific recommendations. Preclinical data and preliminary clinical observations suggest a theoretically major radio-sensitivity and high risk of secondary radio-induced malignancies. It remains discussed whether the knowledge of a germline p53 mutation may influence treatment (trt) choices and have prognostic importance. We reviewed our cohort of BC diagnosed as first tumor in pts with germline p53 mutations within the past 11 years, with an attempt to describe secondary malignancies occurring after trt of primary tumor. Patients and Methods: 9 pts have been diagnosed and treated in our institution within the past 11 years for BC as first tumor in the context of a documented germline p53 mutation. Their tumor characteristics, treatment and follow-up data were extracted from prospectively-registered medical records. Results: Median age at diagnosis of primary BC was 32 (22–48). 7/9 pts had a family history compatible with Li Fraumeni syndrome, while 2 had no family history. No pt had previous knowledge of her p53 mutation. Primary tumors were ductal carcinoma in situ (4), infiltrating ductal carcinoma (4), phyllode tumor (1). 3 pts had conservative surgery and 6/9 underwent mastectomy (M). 6 received loco-regional radiation therapy (RT). None had prophylactic contra lateral mastectomy. Loco-regional (LRR) and contra-lateral relapses are listed in the Table , as well as incidence of second primaries within or outside radiation field. Second primaries were sarcoma in 3/4 cases and 1 was papillary thyroid carcinoma. Conclusions: Because of high risk of second breast primary and probably very high risk of radio-induced breast cancer, BC pts with germline p53 mutations should be advised bilateral prophylactic mastectomy and avoidance of radiation therapy. In this context, the knowledge of the mutation might be of great importance. [Table: see text]

No significant financial relationships to disclose.
Determination of factors predicting for estrogen receptor positive (ER+) breast cancers in BRCA1 mutation carriers
Authors: Comander A. H. , Gallagher B. M. , Krag K. , Wang Y. , Li H. , Gelman R. , Collins L. C. , Schnitt S. J. , Garber J. E. , Tung N.
Published: August 2020

11045

Background: Most breast cancers that occur in BRCA1 mutation carriers tend to lack ER, progesterone receptors and Her2/neu. Foulkes et al.(Clin Can Res 2004; 10: 2029) reported an increase in BRCA1-associated ER+ tumors with increasing age. Little is known about the clinical factors that predict for ER status in this population. Understanding these factors and whether ER+ cancers are sporadic may have important implications for chemoprevention and treatment strategies. Methods: BRCA1mutation carriers who developed an invasive breast cancer from 1973–2008 were identified through the Cancer Risk and Prevention programs at 4 Boston-area hospitals. Clinical characteristics were abstracted from medical and research records. ER status was obtained from pathology reports, and ER staining was repeated when data was missing or when ER was listed as “weak”. Logistic regression was used to model ER positivity for the first invasive breast cancer. Potential predictors included menopausal status, age at cancer diagnosis, prior use of hormone replacement therapy, Ashkenazi-Jewish descent, age at first live birth, tobacco use, and alcohol use. Results: 170 BRCA1 mutation carriers were identified; 56 with ER+ and 114 with ER- first invasive cancers. Menopausal status was found to be a significant predictor of ER status; 18/56 (32%) with ER+ breast cancer were postmenopausal vs.14/114 (12%) with ER- cancer (odds ratio = 0.30, p = 0.002). Age >= 50 was significant in univariate analysis, 14/ 56 (25%) ER+ vs. 11/ 114 (10%) ER- cancers (p=0.01), but not in multiple regression. Conclusions: While most breast cancers in BRCA1 carriers are ER-, we found postmenopausal BRCA1carriers significantly more likely to develop an ER+ tumor than premenopausal carriers. As mutation carriers are likely to become surgically menopausal before age 50, and menopause and age are correlated, additional data will be needed to sort out the relative contribution of each factor to ER status. An understanding of this issue could have important implications for chemoprevention strategies. Molecular analysis of the ER+ breast cancers in our BRCA1+ cohort is underway to investigate whether ER+ breast cancers are manifestations of the BRCA1 mutation or sporadic.

No significant financial relationships to disclose.
Use of genome-wide scan in women with breast cancer to identify common germline variants that may be associated with recurrence
Authors: Comen E. A. , Lautenberger J. , McGee K. , Kirchhoff T. , Dean M. , Hudis C. , Norton L. , Offit K. , Gold B. , Robson M.
Published: August 2020

11049

Background: Studies of normal DNA variation have identified several common single nucleotide polymorphisms (SNPs) associated with susceptibility to breast cancer. It is not known whether common SNPs are associated with breast cancer outcomes. Methods: Subjects were Ashkenazi Jewish women with familial breast cancer and without BRCA mutations. Subjects were genotyped on an Affymetrix 500K SNP platform in the first phase of a genome-wide association study seeking susceptibility loci (Gold et. al, Proc Natl Acad Sci 2008;105:4340). Clinical variables and outcomes for these women were abstracted from medical records. Using a Cox proportional hazards model, we assessed associations between clinical outcomes and the 111 SNPs that were most significantly associated with susceptibility in the case-control study and heterozygous in the subset of affected patients. Dominant, codominant, and recessive models were explored. A secondary analysis assessed associations with recurrence of 48,562 additional SNPs that were not strongly associated with susceptibility. Results: The 173 subjects were a median of 51 years of age at diagnosis (range 27–74). Median follow-up for surviving subjects was 85.5 months. Most cancers (74.6%) were T1/T2; 62% were N0. Most were ER positive (81.5%), HER2 negative (87.5%). For the entire group, the 5- and 10-year freedom from recurrence was 84% and 72%, respectively. The 5- and 10-year overall survival was 94% and 86%, respectively. Minor alleles at rs6439927 (within CLSTN2,calsyntenin2, a post-synaptic calcium-binding membrane protein, MAF 27%) and rs7943562 ( linked to GALNTL4, an N-acetylgalactosaminyltransferase, MAF 47%) were most strongly associated with disease recurrence (log likelihood ratio chi square P=0.003 and P=0.002, respectively). No additional SNPs from the secondary analysis achieved genome-wide significance. Conclusions: Genome-wide SNP genotyping of women with breast cancer identified two novel loci that may be associated with disease recurrence. Further studies in larger cohorts will be performed to replicate these findings.

No significant financial relationships to disclose.
Effect of the epidermal growth factor receptor antibody panitumumab on triggering of ADCC
Authors: Valerius T. , Dechant M. , Lammerts van Bueren J. J. , Schneider-Merck T. , Berger S. , Beyer T. , Gramatzki M. , Bleeker W. K. , Parren P. W. , van de Winkel J. G.
Published: August 2020

11055

Targeted therapy against the epidermal growth factor receptor (EGF-R) has been introduced into clinical practice for several solid tumors. Thus far, EGF-R- directed antibodies have received approval for the treatment of advanced colorectal and head and neck cancer. Recent studies demonstrated an association between Fcg receptor polymorphisms and clinical outcome. Here, we investigated effector mechanisms of the human IgG2 antibody pantitumumab and zalutumumab—a human IgG1 antibody against EGF-R, currently in phase III clinical evaluation. Both antibodies had similar binding characteristics, but differed in induction of antibody- dependent cellular cytotoxicity (ADCC). Zalutumumab induced potent MNC-mediated ADCC via FcgRIII (CD16), which was poorly triggered by panitumumab. Interestingly, not only zalutumumab but also panitumumab recruited neutrophils for ADCC. Neutrophil- mediated killing is triggered via FcgRIIa (CD32), and is potentially affected by its 131R/H polymorphism. Thus, panitumumab- mediated ADCC was more effective by neutrophils from FcgRIIa-131H homozygous individuals than from -131R individuals. ADCC induced by the IgG1 antibody zalutumumab was not affected by this polymorphism. In an experimental metastasis model in mice, both zalutumumab and panitumumab prevented establishment of metastases at low antibody concentrations which allowed the induction of ADCC but not the inhibition of signalling. Our results may implicate neutrophil- mediated ADCC in the in vivo mechanisms of action of EGF-R- directed antibodies, and suggest that heterogeneity in clinical responses due to the FcgRIIa-131R/H polymorphism can be expected for panitumumab.

[Table: see text]
Correlation of FOXA1 expression with Oncotype Dx recurrence scores
Authors: Ademuyiwa F. O. , Thorat M. , Nakshatri H. , Badve S.
Published: August 2020

11058

Background: Oncotype Dx assay (ODX) is a diagnostic assay that predicts the benefit of chemotherapy and assesses the likelihood of breast cancer (BC) recurrence in patients with estrogen receptor (ER) positive BC. The forkhead-box A1 (FOXA1) transcription factor is linked to ER regulated gene transcription and its expression is associated with ER positive luminal A type BC. Since higher FOXA1 expression is associated with ER positive luminal A BC, we investigated whether FOXA1 expression levels show a correlation with ODX recurrence scores in BC patients. Methods: 120 cases of BC from IU and affiliated hospitals had ODX performed on tumor blocks. 78 patient tumor blocks with ODX recurrence score have been analyzed for FOXA1 using IHC. FOXA1 expression was measured using intensity X percentage scoring. The correlation of FOXA1 scores and ODX recurrence scores with each other and with other variables was examined. Other variables included age, progesterone receptor (PR) status, tumor size, grade, histological type, and race. All p values are two-sided and have been adjusted for multiple comparisons using Bonferroni-Holm's method. Results: Twenty-one of 78 cases analyzed were < 50 years. ODX recurrence scores were low, intermediate, and high in 55.1%, 32.1%, and 12.8% of cases, respectively. Ductal carcinomas accounted for 73.1% of tumors, while 11.5% were lobular. Fifty percent of tumors were histologic grade II, 37% and 13% were grade I and III, respectively. PR was negative in 9 cases. Caucasians accounted for 78.2% of cases vs 7.7% African-Americans. FOXA1 expression correlated negatively with ODX recurrence score (p=.003), and histologic type (p=.024). ODX recurrence score also correlated negatively with PR (p=.034) with 78% of the PR negative cases having a high or intermediate ODX score. The correlation between FOXA1 expression and ODX recurrence score remained significant after adjusting for multiple comparisons and controlling for confounders such as histological type, grade, and PR. Conclusions: We demonstrate that FOXA1 expression has an inverse correlation with ODX recurrence scores. FOXA1 expression can potentially be used as a prognostic marker in low risk ER positive BC patients who lack access to ODX assays. This observation needs to be confirmed with a larger sample size.

No significant financial relationships to disclose.
FL-DNA approach for noninvasive early diagnosis of colorectal cancer in FOBT-screened patients
Authors: Calistri D. , Rengucci C. , Casadei-Gardini A. , Scarpi E. , Zoli W. , Falcini F. , Milandri C. , Amadori D. , Silvestrini R.
Published: August 2020

11062

Background: A promising approach for the early diagnosis of colorectal cancer (CRC) is the evaluation of genomic DNA integrity (FL-DNA) extracted from stool. Pilot and confirmatory studies carried out by our group have shown that, thanks to its diagnostic accuracy, this molecular assay could be a useful tool for the non-invasive, early diagnosis of CRC. The aim of the present study was to verify whether the FL-DNA method could represent a valid alternative to the fecal occult blood test (FOBT) or whether it could be used alongside FOBT in screening programs to unmask false FOBT positives and spare patients from unnecessary colonoscopy. For this purpose, stool samples were collected from all individuals participating in the FOBT (OC-Sensor, Alpha Wassermann) Regional Screening Program run by the Cancer Prevention Unit of Morgagni-Pierantoni Hospital in Forli. Methods: The program recruited subjects of either sex aged 50 to 69 years. Of the 560 individuals with FOBT-positive stool subjected to colonoscopy, 26 were diagnosed with adenocarcinoma, 264 with high-grade adenoma and 54 with low-grade adenoma. More than one third (216) of the group had only benign disease (hemorrhoids, diverticulitis, inflammation, etc), hyperplastic polyps or nothing. The integrity of fecal DNA was analyzed blindly by the FL-DNA test on the same specimen used for the occult blood determination. Results: Using a cut-off of 10 ng, the molecular analysis detected over 90% of the colorectal cancers and about 50% of the high- and low-grade adenomas. In particular, diagnostic accuracy was similar for lesions of any localization, level of dysplasia and histopathological characteristic, and higher for lesions > 2 cm and multiple lesions. The test also confirmed its capacity to identify colorectal cancer in asymptomatic individuals. Conclusions: FL-DNA represents a valid alternative to or support for approaches currently used in screening programs. A more in depth DNA stool evaluation in negative FOBT individuals could reveal the test's usefulness in unmasking colorectal tumors and adenomas missed by FOBT.

No significant financial relationships to disclose.
Evaluation of a gene signature to predict single agent dacetuzumab (SGN-40) activity in patients with DLBCL
Authors: Advani R. , Burington B. , Shi X. , de Vos S. , Ansell S. , Forero-Torres A. , Ebens A. , Whiting N. , Dornan D.
Published: August 2020

11063

Background: Dacetuzumab (SGN-40) is a humanized IgG1 monoclonal antibody that binds to CD40, mediates effector cell functions, and activates downstream apoptosis signaling pathways. Dacetuzumab has shown single-agent activity in relapsed/refractory DLBCL in phase I and phase II trials, with multiple objective responses and 1/3 of patients demonstrating tumor shrinkage, defined as a decrease in tumor volume (SPD) of at least 10%. We previously reported a 14-gene signature (ASH 2008 #1593) that was strongly associated with dacetuzumab sensitivity in DLBCL cell lines. Here, we report an initial evaluation of the gene signature as a classifier of patients likely to demonstrate tumor shrinkage after dacetuzumab therapy. Methods: The original 14 microarray probes were chosen for high correlation with in vitro dacetuzumab sensitivity (IC25) in 31 NHL cell line models. Matching qRT-PCR probes were developed and confirmed to correlate with the microarray probes in paired cell line samples. In this retrospective analysis, archived paraffin blocks from a 26 patient subset of the phase I and II trials, with a diagnosis of DLBCL and available tumor measurements, were assayed by qRT-PCR. Results: Overall, 42% of patients (11/26) exhibited decreased SPD of at least 10%. Of those who were marker +, 10 out of 13 (78%) had 10% or better decreases in SPD, whereas only 1 of 13 patients who were marker - demonstrated tumor shrinkage (8%). The overall accuracy for predicting tumor shrinkage was 85% (one-sided P=0.002, by permutation test). Among the 14 genes contributing to the multivariate signature, CD22 and VNN2 were the most strongly down-regulated in specimens from patients without at least a 10% decrease in SPD (P=0.14 and P=0.10, respectively), while IGF1R and CTSC were the most strongly up-regulated (P=0.05 and P=0.08, respectively). Conclusions: A 14-gene signature appears to predict tumor shrinkage in DLBCL patients receiving dacetuzumab in single-agent clinical trials (P=0.002). A larger clinical data set will be analyzed to further evaluate the correlation of this gene signature with objective clinical response rates..

[Table: see text]
Circulating tumor cells (CTCs) and endothelial cells (CECs) in primary lung cancer
Authors: Tanaka F. , Yoneda K. , Hashimoto M. , Takuwa T. , Matsumoto S. , Okumura Y. , Kondo N. , Hasegawa S. , Fukuoka K. , Nakano T.
Published: August 2020

11066

Background: Circulating tumor cell (CTC), a surrogate of distant metastasis, and circulating endothelia cell (CEC), a surrogate of angiogenesis, are potentially useful in the diagnosis of malignant tumors, but clinical significance of CTC/CEC in primary lung cancer (LC) remains unclear. Methods: A total of 342 cases who presented at our institute from August 2007 through December 2008 were included in the study. CTC/CEC in the peripheral blood (7.5mL/4.0mL) were captured and quantitatively evaluated with the “CellSearch” system without knowledge of clinical characteristics of each case. Results: In 42 (30.7%) of 137 LC cases, CTC in the peripheral blood was positive (CTC-count, more than 1 cell/7.5mL), and the maximum CTC-count was 62 cells. In 11 (18.3%) of 145 cases with non-malignant (NM) diseases, CTC was also positive; however, in NM cases, CTC-count was 1 (cell/7.5mL) in most CTC-positive cases and the maximun CTC-count was 2. Among LC cases, the incidence of case with CTC-positive (CTC-count, 1 or more) was highest in small cell carcinoma cases (7/10, 70.0%), followed by squamous cell carcinoma (9/22, 40.9%) and adenocarcinoma (23/94, 24.5%) cases; the incidence of CTC-positive case was significantly higher in stage IV cases (68.6%; p<0.001), but it should be noted that CTC was positive in 17.4% of stage I cases and 15.4% of stage II cases. The mean CEC number (/4.0mL) was significantly higher in LC cases than in NM cases (97.5 versus 52.5, respectively; p=0.023), Among LC cases, the mean CEC significantly increased along with tumor progression (mean CEC for stage I, II, III, and IV cases: 58.7, 57.9, 83.4, and 178.4, respectively; p=0.002). Conclusions: CTC and CEC are useful clinical markers in LC, and its clinical value should be validated in future clinical trials. [Table: see text]

No significant financial relationships to disclose.
Microfluidic device for circulating tumor cell sorting, characterization, and culture
Authors: Saliba A. , Saias L. , Bidard F. , Mathiot C. , Saada V. , Farace F. , Vielh P. , Pierga J. , Viovy J.
Published: August 2020

11068

Background: Circulating tumor cell (CTC) analysis is a promising approach to further characterize the tumor biology and the metastatic process, but has immediate clinical applications such as early predictive markers of response to chemotherapy. However, the devices that are used currently for CTC detection have a poor sensitivity. Microfluidic techniques offer new exciting perspectives in the field of cell sorting for CTC analysis. Methods: We propose here a new microfluidic system and strategy allowing the sorting of cells, their study by confoncal fluorescent microscopy and/or their subsequent culture. The core of our system is a self-organized array of superparamagnetic particles in a microchannel, allowing for the formation of a perfectly periodic and robust array. The superparamagnetic particles are coated with specific antibodies against surface antigens, in order to capture cells according to specific biomarkers. This original cell-separation microchip was integrated into a fully automated platform combining state-of-the-art microfluidic technologies, microvalves and nanofluidic pneumatic pumps. Results: Using B and T lymphocytes mixtures as a model biological system, the capture yield was better than 90%, and the specificity better than 97%. Captured cells were fully viable and were cultured in situ in the magnetic array. In clinics, immunophenotyping of circulating malignant B cells was performed using fluorescent confocal microscopy, and was successfully compared to flow cytometry data for each sample. Results on breast cancer cells will be presented at the meeting. Conclusions: This new system is a promising approach for efficient CTC sorting, analysis and culture in a completely integrated manner.

No significant financial relationships to disclose.
Phase I evaluation of lapatinib (L) and epirubicin (E) in patients (pts) with anthracycline (anth)-naive metastatic breast cancer (MBC)
Authors: O'Connor R. A. , Kennedy M. J. , McDermott S. R. , Tryfanopoulos D. , McCreery C. , Collins D. , Martynyuk O. , Gethins R. , Moulton B. , Crown J.
Published: August 2020

1107

Background: HER-2 alteration confers enhanced anth sensitivity in MBC, although HER-2 is not a target for anth. Topoisomerase 2A (Topo) is a target for anth. Topo amplification has been reported to occur only with HER-2 amplification, in approximately 1/3 of HER-2 amplified BC, providing a rationale for anth + trastuzumab(H) therapy in MBC with co-ampified HER-2/Topo. T+anth is cardiotoxic. L may have similar single agent activity, but less cardiotoxicity than H. L competes with anth for p-glycoprotein and might enhance its activity. The cardiac safety of L + anth needed to be established. Methods: We conducted a phase I evaluation of fixed dose lapatinib (1250 mg/day) with escalating doses of epirubicin (Epi-planned dose levels I-IV :75/80/90/100 mg/m2 ) in pts with HER-2±, anth-naive metastatic BC. Epirubicin was administered q3 weeks. A minimum of 3 (if no dose-limiting toxicity) and a maximum of 6 pts were treated per dose level. Ejection fractions were performed at base line and after every second cycle; BNP and troponin after every cycle. Results: 9 pts were treated, 3 level I, 6 level II. Dose level I was declared the maximum tolerated dose following two occurrences of dose-limiting toxicity in level II. 1 pt experienced febrile neutropenia,1 had > 20% decrease in LVEF. To date there have been 3 PRs by RECIST out of 8 evaluable patients which represents a response rate of 37.5%. Conclusions: Using classic phase I criteria, the combination of L 1250 mg together with Epi 75 mg/m2 combination is relatively well tolerated and suitable for further investigation in a phase II study of HER-2+veTopo+ve MBC patients. We will conduct such a study in pts with HER-2+Topo+ metastatic BC.

[Table: see text]