A phase 1/2 clinical trial of the nitric oxide synthase inhibitor L-NMMA and taxane for treating chemoresistant triple-negative breast cancer.

The inducible nitric oxide signaling (iNOS) pathway is associated with poor prognosis in triple-negative breast cancer (TNBC). Prior studies using in vivo models showed that inhibition of the iNOS signaling pathway using the pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) reduced tumor growth and enhanced survival in patients with TNBC. Here, we report a first-in-class phase 1/2 trial of L-NMMA combined with taxane for treating patients with chemorefractory, locally advanced breast cancer (LABC) or metastatic TNBC. We also examined immune cell correlates of chemotherapy response. 35 patients with metastatic TNBC were recruited: 15 in the phase 1 trial and 24 in the phase 2 trial (including 4 recommended phase 2 dose patients from the phase 1 trial). The overall response rate was 45.8% (11 of 24): 81.8% (9 of 11) for patients with LABC and 15.4% (2 of 13) for patients with metastatic TNBC. Among the patients with LABC, three patients had a pathological complete response at surgery (27.3%). Grade ≥3 toxicity was noted in 21% of patients; however, no adverse events were attributed to L-NMMA. Immune cells analyzed by CyTOF indicated that chemotherapy nonresponders showed greater expression of markers associated with M2 macrophage polarization and increased concentrations of circulating IL-6 and IL-10 cytokines. In contrast, chemotherapy responders showed an increase in CD15+ neutrophils in blood, as well as a decrease in arginase (a marker of protumor N2 neutrophils) in tumor biopsies obtained at the end of treatment. L-NMMA combined with taxane warrants further investigation in larger clinical studies of patients with breast cancer.

ANETT: PhAse II trial of NEoadjuvant TAK-228 plus Tamoxifen in patients with hormone receptor-positive breast cancer.

INTRODUCTION: Neoadjuvant endocrine therapy is often utilized to downstage Estrogen Receptor-positive (ER+) breast cancer prior to surgery. However, this approach is sometimes met with endocrine resistance mechanisms within the tumor. This trial examines the safety and efficacy of tamoxifen in combination with an mTORC1/2 inhibitor, TAK-228, in the neoadjuvant treatment of ER+ breast cancer. METHODS: In this single-arm, open-label trial, pre- and post-menopausal women were enrolled to receive neoadjuvant tamoxifen (20 mg daily) with TAK-228 (30 mg weekly) for 16 weeks prior to surgery. Patient had tissue sampling at baseline, week 6, and week 16. The primary endpoint was change in Ki-67 from baseline to 6 weeks. The toxicity, change in tumor size, pathologic complete response rate, PEPI score, and baseline Oncotype Dx score were also assessed. RESULTS: Twenty-eight women were enrolled on the trial, and 25 completed the entire study course. The combination of tamoxifen and TAK-228 resulted in a significant reduction in Ki-67 from 18.3 to 15.2% (p = 0.0023). The drug was also found to be safe and tolerable. While nausea and hyperglycemia were common side effects, these were manageable. The tumor size also significantly decreased with the treatment, with a median decrease of 0.75 cm (p < 0.0001). There were no pathologic complete responses. CONCLUSION: Tamoxifen and TAK-228 was safe and well tolerated neoadjuvant treatment for ER+ breast cancer, preliminary evidence of activity with significant reduction in both Ki-67 and tumor size, warranting further evaluation in a larger study.

Targeting mTOR and DNA repair pathways in residual triple negative breast cancer post neoadjuvant chemotherapy.

Triple-negative breast cancer (TNBC) patients who do not achieve pathologic complete response post neoadjuvant chemotherapy have a poor prognosis. Alteration in PI3K/mTOR plus DNA repair pathways are some of the major mechanisms of chemotherapy resistance. We designed an open-label phase II clinical trial to evaluate if the combination of everolimus (mTOR inhibitor) plus cisplatin (interferes with DNA function) will improve the rate of pathologic response, as assessed by residual cancer burden (RCB). Twenty-four Stage II/III TNBC patients with residual cancer > 1 cm post neoadjuvant anthracycline and taxane-based chemotherapy were enrolled. Patients received everolimus daily orally at 10 mg for 12 weeks and cisplatin IV at 20 mg/m2 weekly for 4 cycles (21-day cycle), until definitive surgery. The primary endpoint was the rate of RCB-0-I at the surgery. The median age of the whole cohort was 50.1 years, with 66.7% non-Hispanic Caucasians. Of the 24 patients enrolled, 22 were included in the efficacy analysis. Twenty-one patients underwent definitive surgery while one patient developed distant metastasis. Five patients had RCB-I at surgery, a response rate of 23% (5/22). Patients with germline PALB2 mutation or somatic PI3KCA mutation had a pathologic response, achieving RCB-I at the surgery. Three patients had metaplastic histology achieving RCB-I at the surgery. Estimated OS at 1 year was 100% in the RCB-I group vs. 76.5% in others, which was not statistically significant due to the small sample size. Certain cohorts including PALB2 germline mutation carrier and somatic PI3KCA mutations warrant further investigation.Trial registration: Clinicaltrials.gov identifier: NCT01931163. https://clinicaltrials.gov/ct2/show/NCT01931163 .

A randomized, controlled phase II trial of neoadjuvant ado-trastuzumab emtansine, lapatinib, and nab-paclitaxel versus trastuzumab, pertuzumab, and paclitaxel in HER2-positive breast cancer (TEAL study).

BACKGROUND: Neoadjuvant dual human epidermal growth factor receptor (HER2) blockade with trastuzumab and pertuzumab plus paclitaxel leads to an overall pathologic complete response (pCR) rate of 46%. Dual HER2 blockade with ado-trastuzumab emtansine (T-DM1) and lapatinib plus nab-paclitaxel has shown efficacy in patients with metastatic HER2-positive breast cancer. To test neoadjuvant effectiveness of this regimen, an open-label, multicenter, randomized, phase II trial was conducted comparing T-DM1, lapatinib, and nab-paclitaxel with trastuzumab, pertuzumab, and paclitaxel in patients with early-stage HER2-positive breast cancer. METHODS: Stratification by estrogen receptor (ER) status occurred prior to randomization. Patients in the experimental arm received 6 weeks of targeted therapies (T-DM1 and lapatinib) followed by T-DM1 every 3 weeks, lapatinib daily, and nab-paclitaxel weekly for 12 weeks. In the standard arm, patients received 6 weeks of trastuzumab and pertuzumab followed by trastuzumab weekly, pertuzumab every 3 weeks, and paclitaxel weekly for 12 weeks. The primary objective was to evaluate the proportion of patients with residual cancer burden (RCB) 0 or I. Key secondary objectives included pCR rate, safety, and change in tumor size at 6 weeks. Hypothesis-generating correlative assessments were also performed. RESULTS: The 30 evaluable patients were well-balanced in patient and tumor characteristics. The proportion of patients with RCB 0 or I was higher in the experimental arm (100% vs. 62.5% in the standard arm, p = 0.0035). In the ER-positive subset, all patients in the experimental arm achieved RCB 0-I versus 25% in the standard arm (p = 0.0035). Adverse events were similar between the two arms. CONCLUSION: In early-stage HER2-positive breast cancer, the neoadjuvant treatment with T-DM1, lapatinib, and nab-paclitaxel was more effective than the standard treatment, particularly in the ER-positive cohort. TRIAL REGISTRATION: Clinicaltrials.gov NCT02073487 , February 27, 2014.

Spheroid-type of AL amyloid deposition associated with colonic adenocarcinoma: A case report with literature review.

We report a colonic adenocarcinoma associated with diffuse submucosal deposition of a peculiar spheroid-type amyloid identified in the colon, terminal ileum, and appendix. A 65-year-old woman with past medical histories of hypertension, and chronic obstructive pulmonary disease, presented to the emergency room with cramping abdominal pain and nausea. A computed tomography (CT) scan of abdomen showed right colonic volvulus. Emergency right hemicolectomy was performed. The specimen showed colonic adenocarcinoma with focal submucosal invasion (pT1) arising from a villotubular adenoma. A diffuse submucosal spheroid-type amyloid deposition (resembling corpora amylacea-like structures with Liesegang ring formation) was identified in the colon, ileum, and appendix. Electron microscopy examination of this unusual spheroidal-type material further confirmed the presence of amyloid fibrils. Analysis by liquid chromatography-mass spectrometry detected AL (lambda) type amyloidosis in this specimen. Tests for monoclonal gammopathy were not performed because patient consent was not obtained. In tissue section evaluation, however, no plasma cell neoplasm was identified. Cases with isolated AL amyloid deposition in the gastrointestinal tract have been reported rarely, and there is no case report of colonic adenocarcinoma associated with primary amyloid deposition in the English literature.

Effects of Aronia melanocarpa Fruit Juice on Isolated Rat Hepatocytes.

BACKGROUND: Aronia melanocarpa (Michx.) Elliot fruits are very rich in polyphenols - procyanidins, flavonoids, and phenolic acids. OBJECTIVE: On rat hepatocytes, isolated by two-stepped collagenase perfusion, we investigated the effect of A. melanocarpa fruit juice (AMFJ) in two models of liver toxicity caused by (i) metabolic bioactivation of carbon tetrachloride (CCl4), and (ii) tert-butyl hydroperoxide (t-BuOOH)-induced oxidative stress. MATERIALS AND METHODS: Isolated rat hepatocytes are a suitable model for hepatotoxicity studies. We determined the main parameters of the functional and metabolic status of rat hepatocytes: Cell viability (measured by trypan blue exclusion) and the levels of lactate dehydrogenase (LDH), reduced glutathione (GSH), and malondialdehyde (MDA). These parameters were used to investigate the protective effects of AMFJ in the two toxicity models. The effects of AMFJ were compared with those of silymarin. The cells were treated either with AMFJ or silymarin at increasing concentrations of 5 µg/ml, 10 µg/ml, 30 µg/ml, 50 µg/ml, and 100 µg/ml which were used for measuring of IC50. RESULTS: In both toxicity models - CCl4 and t-BuOOH, AMFJ showed statistically significant cytoprotective and antioxidant activities. AMFJ prevented the loss of cell viability and GSH depletion, decreased LDH leakage and MDA production. The effects of AMFJ at the concentrations of 5, 10, 30, and 50 µg/ml were similar to those of the same concentrations of silymarin, while the effect of the highest AMFJ concentration of 100 µg/ml was higher than that of the same silymarin concentration. The effects were concentration-dependent and more prominent in the t-BuOOH model, compared to those in the CCl4 model. CONCLUSION: The cytoprotective and antioxidant effects of AMFJ established in this study might be due to its polyphenolic ingredients, which could influence the cytochrome P450-mediated metabolism of the experimental hepatotoxic substances (CCl4 and t-BuOOH) and could act as free radical scavengers. The stronger effects of the highest AMFJ concentration in comparison with that of silymarin were possibly due to the combined presence of different polyphenols in the juice. SUMMARY: On rat hepatocytes, isolated by two-stepped collagenase perfusion, we investigated the effect of Aronia melanocarpa fruit juice (AMFJ) in two models of liver toxicity caused by i) metabolic bioactivation of carbon tetrachloride (CCl4), and ii) tert-butyl hydroperoxide (t-BuOOH)-induced oxidative stress. In both toxicity models - CCl4 and t-BuOOH, AMFJ showed statistically significant cytoprotective and antioxidant activities. AMFJ prevented the loss of cell viability and GSH depletion, decreased LDH leakage and MDA production. The effects of AMFJ at the concentrations of 5, 10, 30, and 50 µg/ml were similar to those of the same concentrations of silymarin, while the effect of the highest AMFJ concentration of 100 µg/ml was higher than that of the same silymarin concentration. The effects were concentration-dependent and were more prominent in the t-BuOOH model, compared to those in the CCl4 model.

Current knowledge of Aronia melanocarpa as a medicinal plant.

Aronia melanocarpa, native to eastern North America, has become popular in Eastern Europe and Russia. Aronia melanocarpa fruits are one of the richest plant sources of phenolic substances, mainly anthocyanins--glycosides of cyanidin. Anthocyanins are water soluble pigments accounting for the dark blue and even black color of the fruits. Administered orally they can be absorbed as intact glycosides. Aronia melanocarpa fruit juice and anthocyanins derived from the fruits have been studied intensively for the last 15 years. Most of the effects of Aronia melanocarpa anthocyanins are due to their high antioxidative activity. Our investigations have demonstrated a remarkable hepatoprotective, a very good gastroprotective and a pronounced anti-inflammatory effect of Aronia melanocarpa fruit juice in rats as well as a bacteriostatic activity in vitro against Staphylococcus aureus and Escherichia coli and an antiviral activity against type A influenza virus. Research of other authors has demonstrated that Aronia melanocarpa anthocyanins can normalize the carbohydrate metabolism in diabetic patients and in streptozotocin-diabetic rats, have an in vitro antimutagenic activity and exhibit a distinct immunomodulatory activity in human lymphocyte cultures and in patients with breast cancer, suppress the growth of human HT-29 colon cancer cells, inhibit the N-nitrosamine formation in rats and decrease the toxicity and cumulation of cadmium in liver and kidneys. Currently, there are no data in literature about any unwanted and toxic effects of Aronia melanocarpa fruits, juice and extracts.

Protective effect of Aronia melanocarpa fruit juice pretreatment in a model of carbon tetrachloride-induced hepatotoxicity in rats.

UNLABELLED: The main active ingredients of Aronia melanocarpa fruit juice (AMFJ) are phenolic substances, mainly flavonoids from the anthocyanin subclass. AIM: The aim of the present study was to investigate the effect of AMFJ applied as pretreatment in a model of carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. MATERIAL AND METHODS: AMFJ was given orally to rats for 2 days at doses of 5, 10 and 20 ml/kg either alone or as pretreatment before the oral application of CCl4 (0.2 ml/kg, 2 days). The plasma activities of aspartate transaminase (AST) and alanine transaminase (ALT) were measured as markers of the liver cell damage. The levels of malondialdehyde (MDA), a lipid peroxidation marker, were determined in rat liver and plasma. RESULTS: Administration of CCl4 caused an elevation of plasma AST and ALT activities. It also induced an elevation of MDA levels in rat liver and plasma. AMFJ applied alone in the tested doses did not cause any significant changes in the measured enzyme activities and in MDA levels. AMFJ applied as pretreatment prevented the CCl4-induced increase of AST and ALT activities, and also prevented the elevation of plasma and liver MDA levels. CONCLUSIONS: AMFJ showed a protective effect in a model of CCl4-induced hepatotoxicity in rats. This effect might be due to the antioxidant activity of its active ingredients.

Neuropilin-1 suppresses tumorigenic properties in a human pancreatic adenocarcinoma cell line lacking neuropilin-1 coreceptors.

Neuropilin-1 (NRP-1) was first described as a coreceptor implicated in neuronal guidance that bound members of the semaphorin/collapsin family. NRP-1 is also expressed in endothelial cells and is believed to promote angiogenesis by acting as a coreceptor with vascular endothelial growth factor (VEGF) receptor 2. Recent studies suggest that NRP-1 can function through both a VEGF-dependent and VEGF-independent fashion. Expression of NRP-1 has been shown in many human tumors, including pancreatic adenocarcinomas. The exact role of NRP-1 in tumor cells is unknown, particularly in cells that lack the NRP-1 coreceptors VEGF receptor 2 and Plexin-A1. To discern the regulatory role(s) of NRP-1 in pancreatic adenocarcinoma that lack these coreceptors, we overexpressed both full-length NRP-1 and a deletion form of NRP-1 that does not interact with semaphorin or VEGF. Overexpression of either isoform reduced several key tumorigenic properties, including anchorage-independent cell growth and migration in vitro, and resulted in reduced tumor incidence and tumor volume in vivo. Conversely, reduction of NRP-1 expression by small interfering RNA targeting led to enhanced tumor growth. Thus, NRP-1 may play distinct growth regulatory roles in different tumor types, and altering NRP-1 expression or function may be a means of influencing the growth of pancreatic cancers.

Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells.

Vascular endothelial growth factor (VEGF) is associated with tumor angiogenesis and poor prognosis in human colorectal cancer (CRC). VEGF receptor-1 (VEGFR-1 or Flt-1) is a high-affinity receptor for VEGF and is typically considered specific to endothelial cells. Here we report the expression and function of VEGFR-1 in CRC cell lines. VEGFR-1 was expressed in all CRC cell lines studied as determined by RT-PCR, Western blot analysis, FACS, and ELISA. Treatment of the human CRC cell lines HT-29 and SW480 with VEGF-A (a ligand for both VEGFR-1 and -2) or VEGF-B (a ligand specific for VEGFR-1) led to activation of Erk-1/2, SAPK/JNK, and translocation of the p65 subunit of nuclear factor-kappaB into the nucleus. Both VEGF-A and -B led to significant induction of cell motility and invasiveness of CRC cells. Stimulation of cells with VEGF-A or -B also led to larger and more numerous colonies in soft agar. However, activation of VEGFR-1 did not increase CRC cell proliferation. In contrast to the previous paradigm that VEGFRs are not present on tumor cells of epithelial origin, we found that VEGFR-1 is present and functional on CRC cells, and activation by VEGF family ligands can activate processes involved in tumor progression and metastasis.

Protective effect of BP 2-94, a histamine H3-receptor agonist prodrug, in a model of carbon tetrachloride-induced hepatotoxicity in rats.

UNLABELLED: BP 2-94 is a prodrug of the H3-receptor agonist (R)-alpha-methylhistamine [(R)-alpha-MeHA]. BP 2-94 displayed anti-inflammatory, antinociceptive and ulcero-protective properties in experimental animals. AIM: The aim of the present study was to investigate the effect of BP 2-94 in a model of carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. MATERIALS AND METHODS: In order to investigate the effect of BP 2-94 it was applied to rats either alone (20, 40 and 60 micromol kg(-1), 4 days) or as a pretreatment (20, 40 and 60 micromol kg(-1), 4 days) before the application of CCl4 (0,2 ml kg(-1), 2 days). RESULTS: BP 2-94 in the tested doses did not cause significant changes in the plasma aspartate transaminase (AST) and alanine transaminase (ALT) activities and the liver microscopic appearance was normal. Hepatocyte damage, as evident by local areas of liver necrosis and elevated levels of plasma AST and ALT, occurred in rats following acute exposure to CCl4 (0,2 ml kg(-1), 2 days). BP 2-94 applied as a pretreatment dose-dependently reduced the necrotic changes in rat liver and inhibited the increase of plasma AST and ALT activities in response to CCl4. CONCLUSIONS: BP 2-94 had a hepatoprotective effect in a model of CCl4-induced toxicity in rats. This effect might be due the H3-agonistic activity of its active metabolite (R)-alpha-MeHA.

Role of hypoxia-inducible factor 1alpha in gastric cancer cell growth, angiogenesis, and vessel maturation.

BACKGROUND: Hypoxia-inducible factor 1 (HIF-1), a heterodimer comprising the oxygen-regulated subunit, HIF-1alpha, and HIF-1beta, mediates transcription of the gene for vascular endothelial growth factor (VEGF). Overexpression of HIF-1alpha is associated with tumor angiogenesis and tumor cell proliferation and invasion. We examined the effects of inhibiting HIF-1alpha activity on angiogenesis and human gastric cancer growth in vivo. METHODS: Human gastric cancer TMK-1 cells were stably transfected with pHIF-1alphaDN, an expression plasmid encoding a dominant-negative form of HIF-1alpha that dimerizes with endogenous HIF-1beta to produce HIF-1 complexes that cannot activate transcription, or with the empty expression vector (pCEP4). Two clones of pHIF-1alphaDN-transfected cells, DN2 and DN3, were tested in all experiments. We used an enzyme-linked immunosorbent assay to measure VEGF secretion by transfected cells cultured in hypoxic (1% O2) or nonhypoxic (20% O2) conditions. We used subcutaneous and orthotopic mouse tumor models to examine the growth of tumors derived from injected pHIF-1alphaDN-or pCEP4-transfected cells. Tumor cell proliferation, vessel area (a measure of functional vascular volume), and tumor endothelial cell association with pericyte-like cells (a measure of vessel maturation) were analyzed by immunohistochemical or immunofluorescent staining. All statistical tests were two-sided. RESULTS: DN2 cells and DN3 cells secreted less VEGF than pCEP4-transfected TMK-1 cells when cultured in nonhypoxic or hypoxic conditions (e.g., DN2 versus pCEP4 in nonhypoxic conditions: 645 pg of VEGF/10(6) cells versus 1591 pg of VEGF/10(6) cells, difference = 946 pg of VEGF/10(6) cells [95% confidence interval [CI] = 640 to 1251 pg of VEGF/10(6) cells; P =.006]; DN2 versus pCEP4 in hypoxic conditions: 785 pg of VEGF/10(6) cells versus 2807 pg of VEGF/10(6) cells, difference = 2022 pg of VEGF/10(6) cells [95% CI = 1871 to 2152 pg of VEGF/10(6) cells; P<.001]). In the subcutaneous tumor model, tumors derived from DN2 or DN3 cells had lower final volumes, weights, and vessel areas, less tumor endothelial cell association with desmin-positive cells, and fewer proliferating tumor cells than tumors derived from pCEP4-transfected cells. In the orthotopic tumor model, tumors derived from DN2 cells had smaller volumes and less vessel area and maturation than tumors derived from pCEP4-transfected cells. CONCLUSIONS: Inhibition of HIF-1alpha activity impairs gastric tumor growth, angiogenesis, and vessel maturation.