Lymphovascular Invasion and Perineural Invasion Negatively Impact Overall Survival for Stage II Adenocarcinoma of the Colon.

BACKGROUND: Lymphovascular invasion and perineural invasion are histopathological features associated with higher-risk colon cancer. OBJECTIVE: The purpose of this study was to quantify the impact of lymphovascular and perineural invasion on overall survival after diagnosis and to determine the protective effect of adjuvant chemotherapy for early adenocarcinoma with high-risk factors. DESIGN: This was a retrospective database review of the 2010-2014 National Cancer Database for colon cancer. SETTINGS: Individuals diagnosed with invasive adenocarcinoma of the colon (histology code 8140) with primary surgical resection with >12 nodes harvested and no positive nodes on pathological examination were included. PATIENTS: A total of 32,493 patients underwent surgical resection for stage II adenocarcinoma of the colon. INTERVENTIONS: The study involved multivariate Cox regression analysis of the impact of lymphovascular and perineural invasion and adjuvant chemotherapy on overall survival after a diagnosis of stage II adenocarcinoma of the colon. MAIN OUTCOME MEASURES: Survival after a diagnosis of stage II adenocarcinoma of the colon was measured. RESULTS: Five-year survival after diagnosis and surgical resection without adjuvant chemotherapy was lower for patients with lymphovascular (60.0%), perineural (56.9%), and lymphovascular and perineural invasion (55.8%) compared with double-negative disease (66.1%). Log-rank testing confirmed that adjuvant chemotherapy improved 5-year survival after diagnosis for lymphovascular (85.5%), perineural (83.6%), and lymphovascular and perineural invasion (74.3%). After controlling for differences in cohorts, Cox regression analysis showed an increased HR for mortality of 14.0% for lymphovascular (HR = 1.141 (95% CI, 1.060-1.228)), 32.1% for perineural (HR = 1.321 (95% CI, 1.176-1.483)), and 41.0% for lymphovascular and perineural invasion (HR = 1.409 (95% CI, 1.231-1.612)) compared with having neither. Chemotherapy showed a 43% reduction in hazard for mortality (HR = 0.570 (95% CI, 0.513-0.633)). LIMITATIONS: The study was limited by its retrospective review and observational bias. CONCLUSIONS: Lymphovascular and perineural invasion have a detrimental effect on survival after diagnosis of stage II adenocarcinoma of the colon. Chemotherapy may be protective specifically when lymphovascular and perineural invasion are present. See Video Abstract at http://links.lww.com/DCR/A786.

Myelodysplastic Syndromes Over Time: A Comparison of Two Patient Cohorts.

BACKGROUND: With advances in myelodysplastic syndromes (MDS), patient cohorts from different time periods might be different. OBJECTIVES: To compare presentation and outcomes between two cohorts. METHODS: Data were collected from George Washington University Medical Center, Washington, DC, USA 1986-1987 (DC), and Tel Aviv Medical Center, Israel 1999-2009 (TA). RESULTS: The study comprised 227 patients (139 TA, 88 DC). TA patients were older (75.4 ± 9.8 vs. 63.8 ± 14.3 years, P < 0.001) and had more cardiovascular diseases (56.8% vs. 14.8%, P < 0.001), fewer cytopenias (1.67 ± 0.82 vs. 2.0 ± 0.93, P = 0.003), and lower mean corpuscular volume (94.3 ± 9.9 fl vs. 100.5 ± 15.3 fl, P < 0.001). Hemoglobin, leukocyte, neutrophil, and platelet counts were similar. More TA patients had dysplasias. Bone marrow cellularity and cytogenetics were similar, but more TA patients had blasts < 5% (73.4% vs. 50.6%, P = 0.003). More TA patients had early French-American-British (FAB) disease (66.9% vs. 40.9%, P < 0.001) and lower risk disease per International Prognostic Scoring System (81% vs. 50%, P < 0.001). The 5 year survival (5YS) of TA patients was not significantly greater (62% vs. 55%). 5YS by FAB was also slightly greater for TA patients (77% vs. 65% for early FAB; 43% vs. 37% for advanced FAB, P > 0.05). CONCLUSIONS: Although patients diagnosed with MDS at a later period were older and had more cardiovascular co-morbidities, they had fewer cytopenias, tended to have earlier disease, and had minimally greater, but not significant, 5YS.

Phase I/II trial assessing bendamustine plus bortezomib combination therapy for the treatment of patients with relapsed or refractory multiple myeloma.

Bendamustine, active in multiple myeloma (MM), is a bifunctional mechlorethamine derivative with alkylating properties. Bortezomib, approved to treat MM, is effective in combination with alkylators. The tolerability and efficacy of bendamustine plus bortezomib in relapsed/refractory MM was assessed in an open-label, dose-escalating, phase I/II study. Patients aged ≥18 years received intravenous bendamustine 50, 70, or 90 mg/m(2) (days 1 and 4) plus bortezomib 1·0 mg/m(2) (days 1, 4, 8, and 11) for up to eight 28-day cycles. No dose-limiting toxicity was observed after cycle 1; bendamustine 90 mg/m(2) plus bortezomib 1·0 mg/m(2) was designated the maximum tolerated dose (MTD). The most common grade 3/4 adverse events were leucopenia (58%), neutropenia (50%), lymphopenia (45%), and thrombocytopenia (30%). Primary efficacy measure was overall response rate (ORR), which was the combined complete response (CR), very good partial response (VGPR), partial response (PR), and minimal response (MR). ORR was 48% (one CR, two VGPR, nine PR, and seven MR) for all 40 enrolled patients, 52% (16/31) at the MTD (90 mg/m(2) ), and 42% and 46% for prior use of bortezomib (n = 31) or alkylators (n = 28) respectively. Bendamustine plus bortezomib was well tolerated with promising efficacy in this heavily pretreated population.

Abscisic acid and CO2 signalling via calcium sensitivity priming in guard cells, new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses.

BACKGROUND: Stomatal guard cells are the regulators of gas exchange between plants and the atmosphere. Ca(2+)-dependent and Ca(2+)-independent mechanisms function in these responses. Key stomatal regulation mechanisms, including plasma membrane and vacuolar ion channels have been identified and are regulated by the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). SCOPE: Here we show that CO(2)-induced stomatal closing is strongly impaired under conditions that prevent intracellular Ca(2+) elevations. Moreover, Ca(2+) oscillation-induced stomatal closing is partially impaired in knock-out mutations in several guard cell-expressed Ca(2+)-dependent protein kinases (CDPKs) here, including the cpk4cpk11 double and cpk10 mutants; however, abscisic acid-regulated stomatal movements remain relatively intact in the cpk4cpk11 and cpk10 mutants. We further discuss diverse studies of Ca(2+) signalling in guard cells, discuss apparent peculiarities, and pose novel open questions. The recently proposed Ca(2+) sensitivity priming model could account for many of the findings in the field. Recent research shows that the stomatal closing stimuli abscisic acid and CO(2) enhance the sensitivity of stomatal closing mechanisms to intracellular Ca(2+), which has been termed 'calcium sensitivity priming'. The genome of the reference plant Arabidopsis thaliana encodes for over 250 Ca(2+)-sensing proteins, giving rise to the question, how can specificity in Ca(2+) responses be achieved? Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca(2+) signal transduction, a topic of central interest in cell signalling research. In this article we further propose an individual stomatal tracking method for improved analyses of stimulus-regulated stomatal movements in Arabidopsis guard cells that reduces noise and increases fidelity in stimulus-regulated stomatal aperture responses ( Box 1). This method is recommended for stomatal response research, in parallel to previously adopted blind analyses, due to the relatively small and diverse sizes of stomatal apertures in the reference plant Arabidopsis thaliana.

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K channels in Arabidopsis guard cells.

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca2+] ([Ca2+]i), and also on mechanisms that are independent of [Ca2+]i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)-induced stomatal closure response in the [Ca2+]i-elevation-independent pathway? (ii) How do ABA-insensitive mutants affect the [Ca2+]i-elevation-independent pathway? (iii) Does ABA enhance (prime) the Ca2+ sensitivity of anion and inward-rectifying K+ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca2+]i elevations and clamping [Ca2+]i to resting levels. The absence of [Ca2+]i elevations was confirmed by ratiometric [Ca2+]i imaging experiments. ABA-induced stomatal closure in the absence of [Ca2+]i elevations above the physiological resting [Ca2+]i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca2+]i elevations. The ABA-insensitive mutants ost1-2, abi2-1 and gca2 showed partial stomatal closure responses that correlate with [Ca2+]i-dependent ABA signaling. Interestingly, patch-clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca2+ to activate S-type anion channels and down-regulate inward-rectifying K+ channels, providing strong evidence for a Ca2+ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca2+]i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca2+]i sensitivity of stomatal closure mechanisms.

Guard cell ABA and CO2 signaling network updates and Ca2+ sensor priming hypothesis.

Stomatal pores in the epidermis of plants enable gas exchange between plants and the atmosphere, a process vital to plant life. Pairs of specialized guard cells surround and control stomatal apertures. Stomatal closing is induced by abscisic acid (ABA) and elevated CO(2) concentrations. Recent advances have been made in understanding ABA signaling and in characterizing CO(2) transduction mechanisms and CO(2) signaling mutants. In addition, models of Ca(2+)-dependent and Ca(2+)-independent signaling in guard cells have been developed and a new hypothesis has been formed in which physiological stimuli are proposed to prime Ca(2+) sensors, thus enabling specificity in Ca(2+)-dependent signal transduction.

Neoadjuvant chemotherapy and transoral surgery as a definitive treatment for oropharyngeal cancer: A feasible novel approach.

BACKGROUND: The purpose of this study was to present our evaluation of the outcome of oropharyngeal cancer managed with neoadjuvant chemotherapy and transoral surgery (TOS) with neck dissection as definitive treatment. METHODS: This is a case series of 17 patients with advanced oropharyngeal cancer who were treated with neoadjuvant chemotherapy followed by TOS. The treatment details and oncologic outcome are reported. The volumetric response of the tumor to neoadjuvant chemotherapy is evaluated and validated by histopathology. RESULTS: Seventeen patients with TNM stages III and IV oropharyngeal cancer constitute this series for survival analysis. On a median and mean follow-up of 31 and 40 months, respectively, 16 of the 17 patients were alive without recurrence. Disease-specific survival (DSS) and overall survival (OS) at 3 years were 94.1%. CONCLUSION: Adjuvant chemotherapy followed by TOS and neck dissection is a feasible and efficacious novel therapeutic approach for definitive management of moderately advanced oropharyngeal cancer, reserving radiotherapy (RT) for salvage or adverse features. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1837-1846, 2016.

A Point Mutation in DNA Polymerase ß (POLB) Gene Is Associated with Increased Progesterone Receptor (PR) Expression and Intraperitoneal Metastasis in Gastric Cancer.

Increased expression of progesterone receptor (PR) has been reported in gastric cancer (GC). We have previously identified a functional T889C point mutation in DNA polymerase beta (POLB), a DNA repair gene in GC. To provide a detailed analysis of molecular changes associated with the mutation, human cDNA microarrays focusing on 18 signal transduction pathways were used to analyze differential gene expression profiles between GC tissues with T889C mutant in POLB gene and those with wild type. Among the differentially expressed genes, notably, PR was one of the significantly up-regulated genes in T889C mutant POLB tissues, which were subsequently confirmed in POLB gene transfected AGS cell line. Interestingly, patients with T889C mutation and PR positivity were associated with higher incidence of intraperitoneal metastasis (IM). In vitro studies indicate that PR expression was upregulated in AGS cell line when transfected with T889C mutant expression vector. Cotransfection of T889C mutant allele and PR gene induced cell migration in the cell line. These data demonstrated that T889C mutation-associated PR overexpression results in increased IM. Therefore, T889C mutation-associated PR overexpression may serve as a biomarker for an adverse prognosis for human GC.

miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer.

MicroRNA (miRNA) dysfunction is associated with a variety of human diseases, including cancer. Our previous study showed that miR-671-5p was deregulated throughout breast cancer progression. Here, we report for the first time that miR-671-5p is a tumor-suppressor miRNA in breast tumorigenesis. We found that expression of miR-671-5p was decreased significantly in invasive ductal carcinoma (IDC) compared to normal in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. cDNA microarray data revealed that differentially expressed genes when miR-671-5p was transfected are associated with cell proliferation, invasion, cell cycle, and EMT. These data indicate that miR-671-5p functions as a tumor suppressor miRNA in breast cancer by directly targeting FOXM1. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management.

Dependable social relationships predict overall survival in Stages II and III breast carcinoma patients.

OBJECTIVE: The effect of support, from dependable non-household relationships, on breast cancer progression was studied using a prospective, longitudinal design. METHODS: Dependable social support was assessed in women with invasive breast carcinoma Stages II and III within 18 months after diagnosis. Disease outcome was monitored for 8 to 9 years. Cox regression analyses, including the Nottingham Prognostic Index (NPI) score of disease severity, tested the association between the number of dependable support persons and time to death. RESULTS: Ninety participants listed between 1 and 16 dependable nonhousehold support persons (mean = 6, S.D. = 4). There were 21 recurrences and 16 deaths from breast cancer at the end of the study. The number of dependable supports predicted decreased mortality [RR = 0.41 (0.21-0.80), P = .01] after controlling for NPI. NPI predicted increased mortality [RR = 1.6 (1.0, 2.4), P = .05]. CONCLUSIONS: For patients with Stages II and III breast cancer, the number of dependable, nonhousehold relationships predicts decreased mortality, after accounting for disease severity.

Circulating Tumor Cells in Biochemical Recurrence of Prostate Cancer.

OBJECTIVE: Circulating tumor cells (CTCs) have known prognostic implications in metastatic castration-resistant prostate cancer, but little is known regarding its utility in biochemical recurrence (BR) of prostate cancer. The primary objectives were to determine whether CTCs are measurable in patients with BR and whether it can reliably predict prostate-specific antigen (PSA) increase and PSA doubling times (PSADTs). METHODS: BR was identified in patients after prostatectomy or radiation or both, with a PSA increase of ≥ 0.2 for prior prostatectomy or > 2 mg/dL increase for post-nadir in prior radiotherapy. CTCs were enumerated at baseline at the time of study entry using the CellSearch (Janssen Diagnostics, Raritan, NJ) test. RESULTS: The median age for all 36 patients accrued was 69.5 years (range, 51-91) with a median PSA of 1.65 ng/mL (range, 0.2-65.8). Gleason scores ranged from 5 to 9 (median, 7). The majority had prostatectomy (n = 25), external beam radiotherapy (n = 9), CyberKnife (Accuray, Sunnyvale, CA) (n = 1), and combined radiohormonal therapy (n = 1). PSADT ranged from 0.35 to 55 months, with a median of 7.43 months. The incidence of positive CTCs was 8.3% (3 patients), of whom 2 had biopsy-proven bony lesions on presenting with equivocal scans and PSADTs of 2.27 and 3.08 months, respectively. The third CTC-positive patient had a PSADT of 4.99 months. CONCLUSIONS: Obtaining CTCs in unselected patients presenting with BR has a relatively low yield. However, obtaining a positive CTC raises the suspicion of the presence of metastatic disease and may have utility for longitudinal follow-ups of patients with BR.

BP1 Homeoprotein Enhances Metastatic Potential in ER-negative Breast Cancer.

Tumor invasion and metastasis remain a major cause of mortality in breast cancer patients. It was reported that BP1, a homeobox isoform of DLX4, is overexpressed in 80% of breast cancer patients and in 100% of estrogen receptor negative (ER-) tumors. The prevalence of BP1 positive cells and the intensity of BP1 immunoreactivity increased with the extent of ductal proliferation and tumorigenesis. These findings imply that BP1 may play an important role in ER- breast cancer. We sought to determine the effects and mechanisms of BP1 on cell proliferation and metastasis using ER- Hs578T cells as a model. Cells were transfected with either pcDNA3.2 plasmid containing BP1 gene, or pcDNA3.2 vector, then selected and cloned. Overexpression of BP1 increased cell proliferation rate by 2-5 fold (p<0.005), and enhanced the in vitro invasive activity by 25-65 fold (p<0.001). Microarray experiments were performed to identify differentially expressed genes when BP1 is overexpressed. The gene expression profile of the transfected cell lines were compared, resulting in 71 differentially expressed genes with a fold-change of >=2.0. Of those genes, 49 were up-regulated and 22 were down-regulated. Significant pathways were identified involving cell proliferation and metastasis. These data demonstrated that overexpression of BP1 significantly enhanced cell proliferation and metastatic potential in ER- Hs578T cells. Further analysis with more ER- cell lines and patient samples is warranted to establish BP1 as a therapeutic target for ER- breast cancer.

BP1, an isoform of DLX4 homeoprotein, negatively regulates BRCA1 in sporadic breast cancer.

INTRODUCTION: Several lines of evidence point to an important role for BP1, an isoform of DLX4 homeobox gene, in breast carcinogenesis and progression. BRCA1 is a well-known player in the etiology of breast cancer. While familial breast cancer is often marked by BRCA1 mutation and subsequent loss of heterozygosity, sporadic breast cancers exhibit reduced expression of wild type BRCA1, and loss of BRCA1 expression may result in tumor development and progression. METHODS: The Cister algorithm and Genomatix program were used to identify potential BP1 binding sites in BRCA1 gene. Real-time PCR, Western blot and immunohistochemistry analysis were performed to verify the expression of BRCA1 and BP1 in cell lines and breast cancer tissues. Double-stranded siRNA transfection was carried out for silencing BP1 expression. ChIP and EMSA were used to confirm that BP1 specifically binds to BRCA1. RESULTS: A putative BP1 binding site was identified in the first intron of BRCA1, which was confirmed by chromatin immunoprecipiation and electrophoresis mobility shift assay. BP1 and BRCA1 expression were inversely correlated in breast cancer cell lines and tissues, suggesting that BP1 may suppress BRCA1 transcription through consensus sequence binding. CONCLUSIONS: BP1 homeoprotein represses BRCA1 expression through direct binding to its first intron, which is consistent with a previous study which identified a novel transcriptional repressor element located more than 500 base pairs into the first intron of BRCA1, suggesting that the first intron plays an important role in the negative regulation of BRCA1. Although further functional studies are necessary to confirm its repressor activity towards BRCA1, the elucidation of the role of BP1 in breast tumorigenesis holds great promise in establishing BP1 as a novel target for drug therapy.

Isolation of a strong Arabidopsis guard cell promoter and its potential as a research tool.

BACKGROUND: A common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in Arabidopsis guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter. An additional drawback of the 35S promoter is that ectopically expressing a gene throughout the organism could cause pleiotropic effects. To improve available methods for targeted gene expression in guard cells, we isolated strong guard cell promoter candidates based on new guard cell-specific microarray analyses of 23,000 genes that are made available together with this report. RESULTS: A promoter, pGC1(At1g22690), drove strong and relatively specific reporter gene expression in guard cells including GUS (beta-glucuronidase) and yellow cameleon YC3.60 (GFP-based calcium FRET reporter). Reporter gene expression was weaker in immature guard cells. The expression of YC3.60 was sufficiently strong to image intracellular Ca2+ dynamics in guard cells of intact plants and resolved spontaneous calcium transients in guard cells. The GC1 promoter also mediated strong reporter expression in clustered stomata in the stomatal development mutant too-many-mouths (tmm). Furthermore, the same promoter::reporter constructs also drove guard cell specific reporter expression in tobacco, illustrating the potential of this promoter as a method for high level expression in guard cells. A serial deletion of the promoter defined a guard cell expression promoter region. In addition, anti-sense repression using pGC1 was powerful for reducing specific GFP gene expression in guard cells while expression in leaf epidermal cells was not repressed, demonstrating strong cell-type preferential gene repression. CONCLUSION: The pGC1 promoter described here drives strong reporter expression in guard cells of Arabidopsis and tobacco plants. It provides a potent research tool for targeted guard cell expression or gene silencing. It is also applicable to reduce specific gene expression in guard cells, providing a method for circumvention of limitations arising from genetic redundancy and lethality. These advances could be very useful for manipulating signaling pathways in guard cells and modifying plant performance under stress conditions. In addition, new guard cell and mesophyll cell-specific 23,000 gene microarray data are made publicly available here.