bluuebunny/crossref_metadata_2025 huggingface dataset:
Trastuzumab treatment and the risk of central nervous system (CNS) metastases
Authors: Pinder M. C. , Chang H. , Broglio K. R. , Michaud L. B. , Theriault R. L. , Sahin A. , Buchholz T. , Hortobagyi G. N. , Esteva F. J.
Published: March 2020

1018

Background: In the era of trastuzumab, HER2-positive breast cancer confers an increased risk of central nervous system (CNS) metastases. While several studies have examined CNS metastases in trastuzumab-treated patients, data are sparse regarding CNS metastases in trastuzumab-naïve HER2-positive patients. We evaluated time to CNS metastasis, death, and death subsequent to brain metastasis in relation to trastuzumab treatment. Methods: The study population included 750 patients diagnosed with HER2-positive metastatic breast cancer (HER2+ MBC) between June 1977 and January 2006. The association between trastuzumab treatment and the outcomes of time to CNS metastasis and time to death following CNS metastasis were determined using Cox proportional hazards models that included trastuzumab treatment as a time-dependent covariate. Multivariable Cox proportional hazards models were fit to determine the association between trastuzumab treatment and outcomes after adjustment for known prognostic factors. Patients with HER2+ MBC treated at our institution before trastuzumab was available served as our control group. Results: Of the 750 patients included, 689 patients received trastuzumab during the follow-up period while 61 patients were not treated with trastuzumab. Median follow-up was 32 months. A total of 251 patients developed CNS metastases. After adjusting for other prognostic variables including age, ER status, PR status, pathological stage, and site of initial metastasis, patients who received trastuzumab had 2.84 times the risk of CNS metastases (95 % CI = 1.87, 4.30, p < 0.0001) compared to patients who did not receive trastuzumab. Time to death following brain metastasis did not differ significantly between trastuzumab- treated and -untreated patients. Conclusions: In our large series, patients with HER2+ MBC treated with trastuzumab were at significantly increased risk of developing CNS metastases compared to patients who did not receive trastuzumab. This finding warrants further investigation into biological mechanisms that may account for this difference.

No significant financial relationships to disclose.
BCIRG 007: Serum HER2 levels in women with metastatic HER2-amplified breast cancer
Authors: Valero V. , Roche H. , Pienkowski T. , Canon J. , Zhao Y. , Carney W. , Mackey J. , Taupin H. , Buyse M. , Slamon D.
Published: March 2020

1020

Background: BCIRG 007 is a multicenter, phase 3 randomized trial comparing docetaxel and trastuzumab (TH) with docetaxel, platinum salt (cisplatin or carboplatin) and trastuzumab (TCH) as first line chemotherapy in women with metastatic breast cancer (MBC). Women enrolled in the study had to have primary breast tumors with HER2 amplification as determined by centralized FISH analysis. Methods: We determined the percentage of subjects with HER2 amplification who had elevated (>15ng/mL) baseline levels of serum HER2 prior to the initiation of trastuzumab-based therapies. Baseline was considered to be the last available determination within 21 days prior to first treatment. Results: The median baseline serum HER2 levels was 75.8 ng/mL (range=8–3,280 ng/mL) for all subjects (n=123), with no statistical difference between subjects randomized to receive TH (median=65.9 ng/mL, n=64) and those randomized to receive TCH (median=89.9 ng/mL, n=59). Overall, 89% of the 123 subjects with HER2-amplified primary tumors had serum HER2 levels >15 ng/mL at the time of metastatic disease (86% in TH vs 92% in TCH). Conclusions: There was no statistical impact of baseline serum HER2 levels on any important clinical event: response to treatment, clinical benefit (response or stable disease for more than 24 weeks), disease progression or death. Conversely, when serum HER2 was measured over time, subjects with higher levels had an elevated risk of experiencing progressive disease (p=.003), even after adjustment for extent of disease (1 or 2 vs 3 or more organs involved) and presence of visceral disease. These analyses suggest that monitoring serum HER2 levels over the course of disease may be a means for detecting progressive disease in women with HER2 amplified breast cancer. Given the long intervals between the serum HER2 measurement and progression (up to 9 months), caution is required in interpreting these results.

No significant financial relationships to disclose.
Changes in estrogen receptor (ER), progesterone receptor (PR) and HER2/neu status with time: Discordance rates between primary and metastatic breast pathology samples
Authors: Broom R. J. , Tang P. , Simmons C. , Bordeleau L. , O’Malley F. P. , Miller N. , Andrulis I. L. , Brenner D. M. , Clemons M.
Published: March 2020

1024

Background: The occurrence of changes in tumor receptor profile (ER, PR and HER-2/neu) between primary and metastatic tissue has been recognised and may have significant treatment implications. Previous reports on receptor discordance rates have tended to be from sub-studies of clinical trials. We sought to evaluate the degree of ER, PR and HER-2/neu receptor discordance in patients treated at two Canadian cancer centres. Methods: Archival data was collected on patients for whom tissue from both primary and metastatic sites was available. Patients with recurrences in the breast or axilla were excluded. ER, PR and HER-2/neu status in the primary and metastasis were compared. Additional information including adjuvant therapy and time to metastasis biopsy was gathered. Results: To date, data is available on 80 patients. Hormone receptor (HR) status was available for 96% of primaries and 56% of metastases. HR status on both the primary and the metastasis was performed in 51% of cases. The discordance rate for ER was 21% (2-sided p=0.0096). 12% of patients changed from ER+ to ER- and 9% changed from ER- to ER+. The discordance rate for PR was 37% (2-sided p=0.0001), with all of these patients changing from PR+ to PR-. No significant HR discordance was found among different adjuvant hormone therapy subgroups. There was no correlation between HR discordance and time to rebiopsy. No discordance for HER-2/neu was found. Conclusions: Significant discordance for HR status was observed, 21% for ER and 37% for PR. HER-2/neu discordance was not observed. We are currently exploring the practical implications of these discordance rates on the management of advanced breast cancer patients in a prospective trial.

No significant financial relationships to disclose.
Final Results of ECOG1100: A phase I/II study of combined blockade of the ErbB receptor network in patients with HER2- overexpressing metastatic breast cancer (MBC)
Authors: Moulder S. L. , O’Neill A. , Arteaga C. , Pins M. , Sparano J. , Sledge G. , Davidson N.
Published: March 2020

1033

Background: Activation of EGF receptor has been associated with resistance to trastuzumab in breast cancer cell lines. EGFR tyrosine kinase inhibitors inhibit HER2 phosphorylation and synergize with trastuzumab in HER2+ cell lines that co-express EGFR. Methods: Pts with MBC and HER2 overexpression by immunohistochemistry (3+) and/or HER2 gene-amplification by FISH, 0–2 prior chemotherapy regimens for met disease, LVEF 50%, and no prior trastuzumab were treated with trastuzumab 2 mg/kg/wk and gefitinib 250- 500 mg/day until disease progression, unacceptable toxicity or withdrawal of consent. The phase I portion of the trial used a 3+3 design to determine MTD. In the phase II portion of the trial, patients were stratified based upon prior chemotherapy exposure (Group 1= no prior exposure to chemotherapy, Group 2= prior exposure to 1–2 chemotherapy regimens). Response measured using RECIST criteria. The primary endpoint was to increase proportion progression free from 50 to 65% at 6 months in Group 1 and from 50 to 70% at 3 months in Group 2. Results: Phase I: DLT (Grade 3 diarrhea) occurred in 2/3 patients treated at the 500 mg/day dose level of gefitinib in combination with weekly trastuzumab. 0/3 patients treated at the 250 mg/day dose level experienced DLT. This was considered MTD and was the dose selected for the Phase II portion of the trial. Phase II: 36 eligible pts were enrolled. Most patients were ECOG PS of 0 and had visceral organ involvement. Of the patients enrolled in Group 1, one pt achieved a CR, one PR and 7 had SD (≥ 24 weeks). Median time to progression (TTP) was 2.9 months (95% CI, 2.5–4). In Group 2 no responses were observed with a median TTP of 2.5 months (95% CI, 1.5- 2.7). Most common severe toxicities were rash (grade 3, 14%) and diarrhea (grade 3, 30%). No grade 3 cardiac toxicity was encountered. Conclusions: Trastuzumab in combination with gefitinib at doses of 250 mg/day demonstrated an acceptable toxicity profile; however, during planned interim analysis, the TTP did not meet predetermined statistical endpoints required for study continuation. These results do not support the further use of this combination and have implications for other trials using trastuzumab and EGFR TK inhibitors simultaneously.

[Table: see text]
Preliminary results of a multicenter phase II trial of vinflunine (with trastuzumab in HER2+ pts) as first-line treatment in metastatic breast cancer
Authors: Peacock N. W. , Spigel D. R. , Mainwaring M. G. , Thompson D. S. , Simons L. , Rubin M. S. , McCleod M. , Harwin W. N. , Schreiber F. J. , Yardley D. A.
Published: March 2020

1043

Background: Vinflunine (VFL) is a new and innovative microtubule inhibitor of the vinca alkaloid class that achieves high intracellular concentrations. By inhibition of tubulin polymerization, cell proliferation is arrested leading to apoptotic death. Demonstrating anti- angiogenic and vascular disrupting activities, VFL has demonstrated significant efficacy as 2nd line chemotherapy in MBC (M. Campone, BJC 2006). This trial was designed to evaluate the response rate and safety of VFL as 1st line therapy in MBC as well as its activity in combination with trastuzumab in HER2+ MBC pts. Methods: Eligibility: 0 prior regimens for MBC, > 6 mo from adjuvant therapy, RECIST measurable disease, ECOG PS 0–2, adequate organ function, < G2 neuropathy. Treatment: 320 mg/m2 IV over 20 minutes q3 weeks; 280 mg/m2 with trastuzumab 6 mg/kg q3 weeks in HER2+ pts. Response evaluations q9 weeks; treatment continued until progression or toxicity. A total of 96 pts will be enrolled, 48 pts per each of 2 cohorts, HER2- and HER2+. Results: 18 pts are enrolled, 13 pts evaluable for toxicity and 12 pts for response. 3 pts received VFL monotherapy and 10 pts were treated with VFL + trastuzumab. Median age: 59 years (43–78). ECOG PS 0: 9 pts, 1: 3 pts, 2: 1 pt. Prior adjuvant chemo: 7 pts (54%), with 5 prior anthracyclines and 6 prior taxanes. 2 pts received adjuvant hormonal therapy only. 4 pts presented with de novo stage IV HER2+ MBC. Metastatic disease sites: liver: 6 pts, lung: 7 pts, bone: 5 pts, lymph nodes: 6 pts. 46% had 3 or more sites of organ involvement. Median of 3 cycles (range:1 - 11) was delivered. 7 pts (58%, all HER2+) had a PR and 4 pts (33%) achieved SD. Only 1 pt progressed. Heme toxicity: G3/4 neutropenia: 2 pts (16%); no febrile neutropenia was noted. G3 non-heme toxicity consisted of N/V: 2 pts and myalgia, 2 pts. There were no G4 events. 4 pts were hospitalized (vomiting: 2, cerebro-vascular accident: 1, back pain: 1 pt). 92% of pts remain free of progression at 6 months. Median TTP has not been reached. Conclusions: Vinflunine is a promising new drug with a high level of activity as first line MBC therapy, especially in combination with trastuzumab. VFL is well tolerated in this patient population with a manageable toxicity profile. Accrual to this trial continues.

[Table: see text]
Phase I study of a novel capecitabine schedule based on Norton-Simon mathematical modeling
Authors: Theodoulou M. , Traina T. A. , Dugan U. , Lake D. , Fornier M. , Feigin K. , Patil S. , Edwards C. , Norton L. , Hudis C. A.
Published: March 2020

1045

Background: We have previously described a mathematical method to optimize chemotherapy dose and schedule (Norton et al, AACR 2005). Capecitabine (C) has activity in breast cancer when conventionally dosed for 14 days (d) q3 weeks (14/7). However, the predicted optimal dosing schedule for C using our model is 7d followed by a 7d rest (biweekly, 7/7). We tested this hypothesis in a Phase I/II study described below. Methods: Eligible patients (pts) have measurable, metastatic breast cancer (MBC), ECOG performance status (PS) =2 and normal organ function. There is no limit to number of prior chemotherapy (CRx) regimens. Pts with prior fluoropyrimidine for MBC are excluded. HER2+ pts must not be candidates for trastuzumab. C is given in divided daily doses for 7d followed by a 7d rest. A standard “3+3” dose escalation scheme employs flat dosing which begins at 1,500mg BID and increases by 500mg/dose level. Primary endpoint is the maximum tolerated dose (MTD), defined as the highest dose for which the incidence of dose-limiting toxicity (DLT) is <33%. Results: 19 pts are now accrued; 17 pts have been treated, 2 withdrew prior to receiving C. Medians: age 47 y (range 34–62 y) and ECOG PS 0 (range 0–2). Sites of MBC: bone 8, viscera 16, soft tissue 11. ER/PR+ 11. HER2+ or unknown 2. Prior adjuvant tx: CRx 17, hormone tx 10. Six pts had adjuvant fluoropyrimidine-based tx. Three pts had 1 prior CRx for MBC; 12 pts received first-line hormone tx for MBC. Fifteen pts had prior anthracycline and taxane. Treatment-related toxicities after a median of 4 cycles (range 1–10) are shown in the table . The MTD has not been reached. Pts continue accrual to the 2500mg/2500mg dose level. Conclusions: Capecitabine 7/7 is well tolerated and allows for safe delivery of higher daily doses than routinely used in practice, as predicted by the mathematical model. Capecitabine 7/7 will be tested in a Phase II program at MSKCC in combination with targeted agents.

[Table: see text]

No significant financial relationships to disclose.
A phase II study of anastrazole and fulvestrant in combination with gefitinib in patients with newly diagnosed ER-positive breast cancer
Authors: Massarweh S. A. , Tham Y. L. , Weiss H. , Fuqua S. , Huang J. , Elledge R.
Published: March 2020

1050

Background: Endocrine therapy for ER+ breast cancer is effective and relatively nontoxic, but is limited by de novo and acquired resistance. Preclinical studies suggest that complete ER blockade along with inhibition of co-expressed growth factor receptors enhances endocrine response and overcomes resistance. Methods: We conducted a phase II trial of combined anastrazole and fulvestrant with gefitinib, an EGFR tyrosine kinase inhibitor, as intial therapy in postmenopausal women with locoregional or metastatic ER+ breast cancer. A tumor core biopsy was done at baseline and after 3 weeks. Patients received treatment for 4 months. Surgery was then offered if the tumor was operable. Primary endpoint was clinical response as defined by RECIST criteria and secondary endpoints were safety/tolerability, complete pathologic response rate (pCR), and biomarker analysis. Planned sample size was 60 patients, but the study closed after 15 patients were enrolled because of poor accrual. Results: Median age at diagnosis was 67 years. Median clinical tumor size was 7 cm and 4 patients had metastases. 11 patients (73%) had ER+/PgR+ tumors and 4 (27%) had ER+/PgR- disease. 3 patients withdraw from the study before response was assessed. Of the12 remaining patients there were 2 complete responses (17%), 5 partial responses (42%), 5 with stable disease (42%), and 2 (17%) with progressive disease. Of the 7 patients who had surgery at 4 months, none had pCR. Most common adverse events were rash in 4 patients, Diarrhea in 4, joint symptoms in 3, and abnormal LFT’s in 3. All adverse events were reversible. Biomarkers, including arrays and Ki67, are pending and will be presented at the meeting. Conclusions: Complete ER blockade using anastrazole and fulvestrant with EGFR inhibition is well-tolerated and has activity in this cohort of patients with relatively advanced ER+ breast cancer. Despite its documented benefit and low toxicity, accrual to neoadjuvant endocrine therapy studies remains poor in the US, largely because of physician hesitance to refer patients for non-chemotherapy studies. More effort is needed to increase physician awareness about the value of endocrine therapy, and to increase accrual to trials that address the question of endocrine resistance.

[Table: see text]
Novel and existing mutations in the EGF receptor tyrosine kinase domain are predictors of optimal resectability (OR) in malignant peritoneal mesothelioma (MPM)
Authors: Foster J. M. , Gatalica Z. , Lilleberg S. , Haynatzki G. , Loggie B. W.
Published: March 2020

10503

Background: MPMs represent 20% of all malignant mesothelioma (MM) cases. The median survival for these tumors is less than a year, and like other peritoneal tumors this is due to intra-abdominal recurrence/progression. Currently there is a paucity of information about the biology of these tumors and molecular perturbations that are involved in tumor formation. Elucidation of mutations and biological pathways active in these tumors may identify valuable prognostic markers, as well as facilitate the development of novel therapies. Methods: Twenty-nine MPM patients were evaluated at a single tertiary center and their tumors were probed for mutations in the catalytic TK domain of EGF receptor (mut+). All specimens were examined for somatic mutations by PCR amplification. Twenty-five patients were treated with cytoreductive surgery (CRS) ± IPHC and complete clinical data including age, sex, cytoreductive score, histology, mutation, and survival was available for comparison of the mut+ and mut- groups. Results: The median age was 56, and 71% of the patients were male with a median follow-up time (MFT) of 14.5 months. Mutations were found in 31% (9/29) of the tumors, 7 of these mutations were novel, and the 8th one was the L858R mutation described in NSCLC. Of the 25 patients managed surgically there were 7 mut+ and 18 mut- ( Table ). OR (=R2a) was achieved in 7/7 (100%) of mut+ group and 9/18 (50%) mut- (p=.03). All mut+ are alive with a MFT of 24 months, while 5/18 (28%) of the mut- group are DOD with a MFT of 7 months (p=.27). In an ANCOVA model OR (p=0.04) was found to be predictive of survival. Conclusions: The EGFR-TK appears to be a common site for mutation in MPM, with the identification of novel and known activation mutation in 31% of tumors. Mut+ was predictive of OR. OR was predictive of survival in short term follow-up, and with longer follow-up, mut+ may be predictive of survival as well as represent a subset of patients who may be responsive to TK-inhibitors therapy.

[Table: see text]

No significant financial relationships to disclose.
TROP2 is a major determinant of growth and metastatic spreading of human cancer
Authors: Alberti S. , Trerotola M. , Vacca G. , Zappacosta R. , Rossi C. , Guerra E. , Bonasera V. , Lasorda R. , Lattanzio R. , Piantelli M.
Published: March 2020

10510

Background: The TROP2 gene encodes a transmembrane calcium signal transducer, involved in the regulation of cell- cell adhesion. Methods: To identify the role of Trop-2 in transformed cells, we studied its expression pattern and function by DNA array and SAGE analysis, Northern and Western blotting, flow cytometry, confocal microscopy and IHC in experimental systems and in man. Overexpression or down-regulation of Trop-2 and directed mutagenesis were used to identify its role in tumor growth and metastasis in vivo. Results: DNA microarray, EST, SAGE, RT-PCR and Northern blot analysis of human tumors revealed expression of the TROP2 gene in most cancers. IHC analysis of human tumors (1755 cases) revealed a corresponding overexpression of Trop-2 protein. Trop-2 potently stimulated the growth of tumor cells, whereas TROP2 siRNA inhibited it. Deletion of the cytoplasmic region of Trop-2 abolished the growth stimulatory capacity, as did mutagenesis of the S303 PKC phosphorylation site. Proteomic analysis showed that multiple PKC isoforms partecipate to the Trop-2 signaling network. In vivo imaging showed dynamic colocalization of PKCs and Trop-2 in vivo in membrane ruffles and podosomes. DN PKCs and siRNA abolished Trop-2-induced growth. Strikingly, comparative global gene expression analysis revealed that TROP2 was the only gene up-regulated across different metastatic models, tumor types and animal species. IHC analysis revealed a dramatic up-regulation in metastases from colon, stomach, breast and ovary tumors in man. To assess if Trop-2 may play a causal role in metastatic spreading, TROP2-transfected KM12SM colon cancer cells were orthotopically injected in nude mice. TROP2-overexpressing transfectants demonstrated increased metastatic potential to the liver. Deletion of the HIKE domain of Trop-2 severely diminished, whereas that of the whole cytoplasmic region vastly increased metastatic diffusion, indicating the existence of both metastatic enhancers and silencers in the Trop-2 cytoplasmic tail. Conclusions: Our findings demonstrate that Trop-2 is a novel, widespread, stimulator of human cancer growth and a unique marker and causal factor of metastatic cancer, and candidate Trop-2 as a target of novel diagnostic and therapeutic procedures.

No significant financial relationships to disclose.
Differential response to primary chemotherapy and long-term survival in patients with triple-negative breast cancer
Authors: Liedtke C. , Mazouni C. , Hess K. R. , Tordai A. , André F. , Symmans W. F. , Gonzalez-Angulo A. M. , Green M. , Hortobagyi G. N. , Pusztai L.
Published: March 2020

10519

Objective. Triple-negative breast cancer is defined as a subtype of invasive breast cancer which lacks estrogen and progesterone receptor expression as well as HER2/neu expression and is highly similar to the basal-like subtype defined by gene expression profiling. Method. 1,143 patients treated at MD Anderson Cancer Center in neoadjuvant trials were included in a retrospective comparative analysis between triple-negative tumors and non-triple-negative tumors for response to neoadjuvant chemotherapy as well as long- term survival. Results. 827/1,143 (72%) patients had received taxanes, either as a single-agent (n=60) or in combination with anthracycline (n=767), whereas the remainder patients received an anthracycline-only chemotherapy. Overall 258/1,143 (23%) tumors were triple- negative. Complete pathological response (pCR) was achieved in 63/257 (25%) patients with triple-negative tumors compared to 99/888 (11%) in patients with non-triple-negative tumors (odds ratio [OR] 1.14, 95%CI: 1.09–1.20, p=.0082). Triple-negative status correlated significantly with high nuclear grade (p<.0001), whereas no significant correlation with any established clinicopathologic parameter was observed. However, 5-year overall survival (5yrOS) was 66% in the triple-negative group compared to 83% in the non-triple-negative control group (OR 2.1, 95%CI: 1.6–2.8, p<.0001). In multivariate analyses, triple-negative status (hazard ratio [HR] 2.0, 95%CI: 1.4–2.8, p<.0001), high nuclear grade, increased tumor size (HR 1.5, 95%CI: 1.3–1.8, p<.0001), positive nodal status (HR 1.4, 95%CI: 1.2–1.7, p=.0002) and high nuclear grade (HR 1.7, 95%CI: 1.1- 2.4, p=.0089) were significantly associated with decreased 5yrOS. When survival was analyzed according to both response rate and triple negative status, achievement of pCR was a stronger predictor of survival compared to triple-negative status. Conclusion. Triple- negative expression status among patients with breast cancer constitutes an independent unfavorable prognostic factor with regards to overall survival unless achieving pCR after neoadjuvant chemotherapy.

No significant financial relationships to disclose.
BRAF mutation predicts MEK-dependence in non-small cell lung cancer (NSCLC)
Authors: Pratilas C. A. , Halilovic E. , Sawai A. , Grbovic-Huezo O. , Rosen N. , Solit D. B.
Published: March 2020

10523

Background: Constitutive ERK signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS and upstream receptors. Missense BRAF kinase domain mutations are frequently observed in melanoma, colon and thyroid cancers and less frequently in lung and other cancer types. The vast majority (>90%) involve a glutamic acid for valine substitution at codon 600 (V600E), which results in elevated BRAF kinase activity. BRAF kinase domain mutations with intermediate and impaired kinase activity have also been identified, most frequently in NSCLC. We have previously reported that tumors with V600E BRAF mutation are selectively sensitive to MEK inhibition. Methods: Using the potent and selective MEK1/2 inhibitor PD0325901 (Pfizer), we examined a panel of NSCLC cell lines with mutant EGFR, KRAS, and/or low, intermediate and high-activity BRAF kinase domain mutations, with regard to MEK dependence. In all but one case, EGFR, KRAS and BRAF mutations were mutually exclusive. Results: Consistent with our prior results, NSCLC cells with V600E BRAF mutation were exquisitely sensitive to MEK inhibition (PD0325901 IC50 of 2nM). The proliferation of cells with non-V600E mutations, including those with high (G469A), intermediate (L597V) and impaired (G466V) kinase activities, was also MEK dependent with IC50's ranging between 2.7 and 80 nM. Inhibition of MEK in these cells resulted in downregulation of cyclin D1 and G1 growth arrest, with variable induction of apoptosis. Despite high basal ERK activity, NSCLC tumor cells with EGFR mutation were uniformly resistant to MEK inhibition (at doses of up to 500nM), despite effective inhibition of ERK phosphorylation. Tumor cells with RAS mutation had a more variable response, with some cell lines demonstrating sensitivity, while others were completely resistant. There was no correlation between basal ERK activity and sensitivity to MEK inhibition. A strong inverse correlation between AKT phosphorylation/ activity and PD0325901 sensitivity was observed. Conclusions: These results suggest that MEK inhibition may be useful therapeutically in tumors with V600E and non-V600E BRAF kinase domain mutations. The results also suggest that inhibition of both MEK and AKT signaling may be required in NSCLC tumors with high basal AKT activity.

No significant financial relationships to disclose.
Microsatellite instability (MSI) due to mutation or epigenetic silencing is associated with increased cytotoxicity to irinotecan (CPT-11) in human colorectal cancer (CRC) cell lines
Authors: Vilar E. , Scaltriti M. , Saura C. , Guzman M. , Macarulla T. , Arribas J. , Tabernero J.
Published: March 2020

10527

Background: MSI is a phenomenon found in tumor DNA of individuals with dysfunction of the mismatch repair system (MMR). Epigenetic inactivation by promoter hypermethylation of hMLH1 causes 15 to 20% of sporadic CRC. In vitro studies have suggested an increased sensitivity to CPT-11 of MMR-deficient human CRC cell lines. Chemosensitivity evaluation in preclinical models of human CRC cell lines according to the MMR status could help in the design of specific studies in the clinical setting. Methods: We have performed drug cytotoxicity assays to compare sensitivity to CPT-11 in several human CRC cell lines with different MMR gene status that resemble the most common clinical situations in CRC patients. HCT116, HCT15, SW48 and RKO are MSI-High (MSI-H), being HCT116 due to a homozygous nonsense mutation of hMLH1 gene, HCT15 due to MSH6 mutation (both of them similar to hereditary cases) and both SW48 and RKO due to methylation of hMLH1 promoter (as MSI-H sporadic CRC cases). HT29 expresses normal levels of MMR proteins (as microsatellite stable (MSS) CRC cases). Drug concentrations resulting in 50% growth inhibition (IC50) were determined by a curve-fitting analysis and cell cycle analyses in order to characterize the cytotoxicity of cell lines were performed. Results: IC50 values and 95% confidence intervals (CI) are show in Table 1 . hMLH1-deficient cell lines due to either epigenetic silencing or mutation showed very similar IC50 and were 5- to 8-fold more sensitive to CPT-11 than the MSS line. hMSH6- deficient cell line HCT15 has sensitivity closer to MSS than MSI cell lines. Treatment with CPT-11 induced a G2/M arrest. Conclusions: Lack of hMLH1 protein due to either genetic alteration or epigenetic silencing correlates with increased sensitivity to CPT-11. MSI-H CRC cell lines are more sensitive to CPT-11 than MSS. Future clinical trials tailoring chemotherapy regimens based on microsatellite status are warranted.

[Table: see text]

No significant financial relationships to disclose.
Mutations in the proapoptotic BAX gene are associated with defective DNA mismatch repair and altered tumor growth rates in human colon cancers
Authors: Sinicrope F. , Rego R. L. , French A. J. , Foster N. R. , Sargent D. J. , Windschitl H. , O'Connell M. J. , Thibodeau S. N.
Published: March 2020

10529

Background: BAX mutations are associated with defective DNA mismatch repair (MMR) in human colon cancers. However, the impact of BAX inactivation upon tumor cell apoptosis and proliferation in vivo remain unknown. We analyzed and compared caspase-3 and Ki-67 expression in tumors with and without BAX mutations. Methods: TNM stage II and III (n= 377) colon carcinomas were studied from participants in a 5-FU-based adjuvant therapy trial. Archival tumors were analyzed for instability at the BAT26 mononucleotide locus using polymerase chain reaction and hMLH1, hMSH2 and hMSH6 by immunohistochemistry (IHC). Frameshift mutations in a tract of eight deoxyguanosines within BAX were analyzed. Expression of caspase-3 and Ki-67 proteins were analyzed by IHC. Results: Thirty-nine of 377 (10%) tumors showed defective MMR defined as instability at BAT26 and loss of either hMLH1, hMSH2 and/or hMSH6 proteins. BAX mutations were found in 20 of 37 (54%) MMR deficient tumors and in 1 of 50 (2%) tumors with intact MMR. Mean and median number of caspase-3-positive cells were increased in tumors with defective MMR (p= 0.04), but did not differ based upon BAX status [ Table ]. However, tumors with BAX mutations showed higher Ki-67 labeling indices compared to those with wild type BAX (p= 0.01)[ Table ]. Neither BAX mutations nor caspase-3- positive cells were prognostic in a univariate analysis. Tumors with lower Ki-67 extent had improved overall survival (p=0.06), but not DFS (p=0.24). Defective MMR (vs intact) was associated with better DFS in a multivariate analysis (p= 0.03). Conclusion: MMR deficient colon cancers show frequent BAX inactivation, yet have increased apoptotic rates as indicated by increased caspase-3 expressing tumor cells. BAX mutation was associated with hyperproliferation suggesting a growth advantage compared to wild type tumors.

[Table: see text]

No significant financial relationships to disclose.
The tyrosine kinase src plays a crucial role in leukemic cell proliferation in AML
Authors: Schuch G. , Schäfer E. , Eggert K. , Loges S. , Görn M. , Laack E. , Bokemeyer C. , Fiedler W.
Published: March 2020

10531

Background: Src-family tyrosine kinases are known to be involved in signal transduction pathways of growth factors and cytokines in hematopoetic cells. While the role of other src family members has been studied widely, only few data exist about c-src in leukemia. The actual study was performed to analyze src mutations in leukemic cells and to determine the role of pp60src in leukemic cell proliferation. Methods: AML cell lines and primary samples were analyzed for expression and activation of src by RT-PCR and Western blot analyses. Mutational analyses were performed by sequencing of C-terminal cDNA from 60 AML samples. The effects of src inhibition were studied by src-specific inhibitors PP1 and PP2 or by siRNA transfection. Effetcs of src inhibition were monitored in proliferation assays and analyzes of signalling through Erk1/2 and apoptosis by annexin V staining and DNA laddering. Results: In all 60 patients analyzed expression of c-src mRNA was detected by RT-PCR. Western blot analyses confirmed strong expression of src on the protein level and revealed a robust activation of the protein as determined by tyrosine phosphorylation. Incubation of leukemic cells with PP1 and PP2 caused significant inhibition of proliferation in a dose dependent manner. Similar results were observed after transfection with specific siRNAs. Src inhibition blocked phosphorylation of pp60src, Erk1/2 and induced apoptosis in leukemic cells. Mutational analyses as performed by SSCP/heteroduplex and bi-directionally sequencing revealed wildtype sequence in cell lines and 60 clinical samples. Conclusions: In summary, pp60src is highly expressed and activated in cell lines and clinical samples of human AML. Moreover, phosphorylation of src is essential for leukemic cell proliferation. Underlying mutations in the coding sequence of c-src causing constitutive activation could be excluded. These data suggest that pp60src plays a crucial role in AML and src inhibition by targeted therapy might offer a useful new approach in the treatment of AML.

No significant financial relationships to disclose.
Clinical relevance of circulating CK-19 mRNA-positive cells detected during the period of adjuvant tamoxifen treatment in patients with early breast cancer
Authors: Xenidis N. , Markos V. , Apostolaki S. , Perraki M. , Pallis A. , Sfakiotaki G. , Kafousi M. , Stathopoulos E. , Kakolyris S. , Mavroudis D. , Georgoulias V.
Published: March 2020

10534

Background: To evaluate the effect of adjuvant treatment with tamoxifen on the CK-19 mRNA+ circulating tumor cells (CTCs) in patients with early-stage breast cancer. Methods: CTCs were prospectively and longitudinally detected using a specific real-time PCR for CK-19 mRNA in 119 patients with estrogen and/or progesterone receptor-positive tumors during the period of tamoxifen administration. Results: Twenty-two (18.5%) patients had detectable CK-19 mRNA+ CTCs after the completion of adjuvant chemotherapy and in 15 (68.2%) of them adjuvant tamoxifen could not eliminate these cells (persistently positive). In 68 (57.1%) patients no CK-19 mRNA+ CTCs could be detected throughout the follow up period (persistently negative). Seven (46.7%) of the 15 persistently positive and six (8.8%) of the 68 persistently negative patients developed disease recurrence (p=0.00026). Persistence of CK-19 mRNA+ CTCs was associated with a significantly shorter median disease-free interval (p=0.0001) and overall survival (0.0005). Multivariate analysis revealed that the detection of CK-19 mRNA+ CTCs during the administration of tamoxifen was associated with an increased risk of relapse (HR=22.318, p=0.00006) and death (HR=13.954, p< 0.00001). Conclusions: The detection of CK-19 mRNA+ CTCs throughout the period of adjuvant tamoxifen treatment is an independent poor prognostic factor in patients with early breast cancer.

No significant financial relationships to disclose.
Predicting breast cancer risk in BRCA1 and BRCA2 carriers: Methylation studies using intraductal fluid
Authors: Antill Y. C. , Mitchell G. , Johnson S. A. , Devereux L. , Milner A. , Sridhar A. , Kirk J. A. , Phillips K. , Lindeman G. , Campbell I. G.
Published: March 2020

10537

Background: Genomic alterations are likely to precede the phenotypic changes of breast cancer (BC) and include hyper- methylation of tumour suppressor genes. An intraductal approach, such as ductal lavage (DL), is an attractive means of accessing ductal epithelial and other cells in a prospective fashion. Methods: Women with at least one breast unaffected by BC, with a germline BRCA1 or BRCA2 mutation, attended for 6-monthly DL collection for up to 3 years. Standard methods were used for DNA extraction and bisulfite conversion. Hyper-methylation of p16, RASSF1A, twist and RARβ genes was investigated using a qualitative, real-time, nested PCR assay. Associations between methylation status and categorical variables were tested using Fisher's exact test including cytological findings and BC incidence. Logistic regression was used to examine the relationship between methylation status and patient age, and to examine the effects of multiple variables. Analyses were performed at three levels; samples from a single breast over time and from a single duct over time and each sample in isolation. Results: A total of 173 DL samples from 98 ducts in 56 breasts were analysed in 34 women (16 BRCA1 and 18 BRCA2 mutation carriers) with a median age of 43 years (range 27- 60). DL fluid was collected from all women on at least one occasion (median 2.5 visits, range 1–5). Five women developed BC on study. Methylation of p16 was strongly associated with a known BRCA1 mutation (p=0.0003, p<0.0001 and p<0.0001 at the breast, duct and sample levels respectively) and was also more likely in women with a previous history of contralateral BC (p=0.0008 and p<0.0001 at the duct and sample level respectively). Independent associations were seen for women who developed BC on study and methylation of p16 and RASSF1A (p16: p=0.0023 at the sample level only) and (RASSF1A: p=0.0760, p=0.0178 and p<0.0001- at the breast, duct and sample level respectively). Conclusions: The methylation pattern in a panel of genes could be used as a biomarker for risk of future breast cancer. In addition p16 methylation may be a predictor of BRCA1 mutation status. Further research is required to corroborate these findings as the numbers recruited for this study are small with a short duration of clinical follow up.

No significant financial relationships to disclose.
Detection of distant metastases in breast cancer patients: Comparison between [18F]-fluorodeoxyglucose positron emission tomography and conventional imaging
Authors: Mahner S. , Schirrmacher S. , Müller V. , Habermann C. , Brenner W. , Schwarz J. Dose
Published: March 2020

1054

Background: The presence of distant metastases is a key prognostic factor in breast cancer patients and plays a central role in therapeutic decisions. To detect distant metastases, chest X-ray, abdominal ultrasound and bone scintigraphy are performed as standard of care in many centers. In various tumors, [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) detects metastatic disease with high accuracy, but the diagnostic value in breast cancer still needs to be defined. The aim of this study was to compare the diagnostic performance of FDG-PET with conventional imaging. Methods: In 119 breast cancer patients who presented for restaging, whole-body FDG-PET was compared with chest X-ray, bone scintigraphy and abdominal ultrasound. Each imaging modality was independently assessed and classified as ‘negative’, ‘equivocal’ and ‘positive’ for metastasis. The imaging results were compared with histopathology and clinical follow-up including follow-up imaging procedures which together served as the reference standard. Results: For findings classified as ‘positive’, FDG-PET detected distant metastases with a sensitivity of 87% and a specificity of 83%. In contrast, the sensitivity and specificity of combined conventional imaging procedures was 43% and 98%, respectively. The positive and negative predictive values were 89% and 82% for FDG-PET and 96% and 69% for conventional imaging. Combining ‘equivocal’ and ‘positive’ results, the sensitivity and specificity of FDG-PET was 93% and 76%, respectively, compared to 61% and 86% for conventional imaging. The positive and negative predictive values were 86% and 88% for FDG-PET versus 77% and 75% for conventional imaging. Conclusions: FDG-PET is superior to conventional imaging procedures for detection of distant breast cancer metastases. However, the use of computed tomography (CT), particularly in combined PET/CT, needs to be addressed in future studies comparing anatomical vs. metabolic staging procedures in breast cancer patients.

No significant financial relationships to disclose.
MicroRNAs that underlie response to salvage chemotherapy in ovarian cancer
Authors: Boren T. P. , Cui S. , Chan G. , Dressman H. , Lancaster J. M.
Published: March 2020

10542

Background: MicroRNAs (miRNAs) are non-protein coding, endogenous, small RNAs that can bind complementary mRNA to modulate gene expression and protein translation. Recent studies indicate that miRNAs may be mechanistically involved in ovarian cancer chemosensitivity and may play an important role in the development of chemoresistance. The objective of the current study was to explore the role of miRNAs in the response of platinum-resistant recurrent or persistent ovarian cancer to salvage chemotherapy. Methods: MicroRNA was extracted from 16 ovarian cancer cell lines. All samples were hybridized to a miRNA array that contained Ambion and Invitrogen probe sets. In parallel, the 16 ovarian cancer cell lines were treated with Doxorubicin, Topotecan, Paclitaxel, Docetaxel, and Gemcitabine, and their response to treatment evaluated using proliferation assays. Logistic regression analysis was used to identify miRNAs associated with responsiveness to individual chemotherapeutic agents. Results: Linear regression analysis identified a total of 39 miRNAs significantly associated with salvage chemotherapy responsiveness. Sixteen miRNAs were associated with response to Doxorubicin, 18 associated with response to Topotecan, 2 associated with response to Docetaxel, 9 associated with response to Paclitaxel, and 11 associated with response to Gemcitabine (p<0.05). Ten miRNAs were associated with more than one chemotherapy (miR-126, miR-199a, miR-202 [Doxorubicin, Topotecan, Gemcitabine], miR-26a, miR-320 [Doxorubicin, Topotecan], miR-181b [Doxorubicin, Topotecan, Paclitaxel], 181a [Topotecan, Gemcitabine, Adriamycin, Paclitaxel], miR-99b [Doxorubicin, Paclitaxel, Docetaxel], miR-31 [Docetaxel, Paclitaxel], miR-339 [Doxorubicin, Gemcitabine]). Conclusion: We have identified miRNAs that may influence ovarian cancer responsiveness to salvage chemotherapy. Ten of the miRNAs were associated with chemosensitivity to more than one drug. These miRNAs may regulate common determinants of chemoresistance and may also serve as novel therapeutic targets for patients with chemoresistant ovarian cancer. Further studies are warranted to validate these results.

No significant financial relationships to disclose.
Biomarker analysis by breast cancer phenotype in African-American versus Caucasian patients: Correlates with survival
Authors: Morris G. J. , Topham A. K. , Guiles F. , McCue P. , Schwartz G. F. , Park P. K. , Rosenberg A. L. , Brill K. , Rui H. , Mitchell E. P.
Published: March 2020

10551

Background: Breast carcinomas in African-American (AA) patients (pts) have poorer prognosis and higher likelihood of aggressive basal phenotype (triple negative for ER, PR, HER2) than those in Caucasian (C) patients (Carey et al, JAMA 2006, 295(21):2492; Morris et al, Breast Cancer Res Treat 2006, abstr 3055). We have additionally examined biomarker expression by phenotype in AA pts in our registry to further explain more aggressive behavior in this population. Methods: Stage, grade, ER, PR, Ki-67, HER2, and p53 expressions were compiled for breast carcinomas in 2,230 AA and C pts diagnosed between 1995–2004. Immunohistochemical markers were assayed using antibodies to the above proteins on paraffin-embedded formalin-fixed tissue. Differences in expression were analyzed by Chi- squared and Wilcoxon tests, and survival by Kaplan-Meier estimates. Results: AA pts have higher propensity for basal phenotype breast cancers (20.8% vs 10.4%, p<0.001) and lower propensity for Luminal A/B (ER+/PR+-/HER2-) phenotype (44.2% vs. 54.1%, p<0.001) as compared with C pts. Higher ki-67 proliferation index was found in AA pts (86.4% vs 78.8% in basal, p=0.3423; 37.1% vs. 26.7% in Luminal A/B p=0.0233) as compared with C pts. p53-positivity was higher in AA and C pts in all cases (p=0.0158), higher in AA pts with basal phenotype (p=0.2597), but identical in AA and C pts with luminal phenotypes (p=0.881). Survival was similar in basal phenotypes between races in all cases stage for stage, and controlled for ki-67 and p53 status, with a trend toward poorer survival among luminal phenotypes between races. Conclusions: AA pts have higher propensity for basal phenotype breast cancers than C pts, with higher ki-67 expression in both basal and luminal phenotypes, and higher p53 expression in basal phenotype, but these do not correlate with significant differences in survival by phenotype between races. As neither ki-67 index nor p53 expression can therefore solely explain differences in survival rates seen between races, molecular array studies between races and matched by phenotype are proposed.

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Matrix metalloproteinase 2 (MMP2) polymorphism, Helicobacter pylori (HP) infection and esophageal adenocarcinoma (EA) risk
Authors: Liu G. , Fruh M. , Zhou W. , Zhai R. , Su L. , Heist R. S. , Wain J. C. , Lynch T. J. , Shepherd F. A. , Christiani D. C.
Published: March 2020

10553

Background: Epidemiologic studies suggest that HP infection protects against EA risk, as HP-mediated chronic atrophic gastritis reduces acid reflux. Genetic variations may modify the host response to HP infection and alter this risk. MMP2 expression is commonly upregulated in HP infections. We hypothesize that a functional MMP2 promoter polymorphism (-C1306T) that abolishes a Sp1 binding site and decreases promoter activity will also reduce the protective effects of HP infections in EA risk. Methods: HP status was determined in 98 EA patients and 101 age and gender matched healthy controls, using a commercially available serum immunoblotting kit (Helicoblot 2.1, Genelabs Diagnostics) that measures ever, current, CagA+ and VacA+ HP infections. Genotyping was performed using TaqMan. Data were analyzed using unconditional logistic regression. Results: 39% of cases and 44% of controls (P=0.69) had MMP2 variants (T/T or C/T). 36% of cases and 42% of controls were ever HP infected (P= 0.35). In individuals carrying the MMP2 wild type (CC) genotype, ever HP infection was strongly protective against EA [Odds Ratio (OR) 0.32: 95% CI, 0.13–0.75; P=0.008]. In contrast, in individuals carrying the MMP2 variants that are associated with lower promoter activity, this protective effect was lost (OR 1.76; 95% CI, 0.06–5.2; P= 0.30). Similar results were found when evaluating the MMP2 and current, CagA or VacA infection. Statistical interactions between MMP2 genotype and ever HP infection (P=0.027) and between MMP2 genotype and VacA+ infection (P= 0.035) were significant. Conclusions: We are the first to report that host factors such as the MMP2 polymorphism modulate the role of HP infection in EA susceptibility.

No significant financial relationships to disclose.