Pembrolizumab in microsatellite instability high or mismatch repair deficient cancers: updated analysis from the phase II KEYNOTE-158 study.

BACKGROUND: Pembrolizumab demonstrated durable antitumor activity in 233 patients with previously treated advanced microsatellite instability high (MSI-H) or mismatch repair deficient (dMMR) advanced solid tumors in the phase II multicohort KEYNOTE-158 (NCT02628067) study. Herein, we report safety and efficacy outcomes with longer follow-up for more patients with previously treated advanced MSI-H/dMMR noncolorectal cancers who were included in cohort K of the KEYNOTE-158 (NCT02628067) study. PATIENTS AND METHODS: Eligible patients with previously treated advanced noncolorectal MSI-H/dMMR solid tumors, measurable disease as per RECIST v1.1, and Eastern Cooperative Oncology Group performance status of 0 or 1 received pembrolizumab 200 mg Q3W for 35 cycles or until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR) as per RECIST v1.1 by independent central radiologic review. RESULTS: Three hundred and fifty-one patients with various tumor types were enrolled in KEYNOTE-158 cohort K. The most common tumor types were endometrial (22.5%), gastric (14.5%), and small intestine (7.4%). Median time from first dose to database cut-off (5 October 2020) was 37.5 months (range, 0.2-55.6 months). ORR among 321 patients in the efficacy population (patients who received ≥1 dose of pembrolizumab enrolled ≥6 months before the data cut-off date) was 30.8% [95% confidence interval (CI) 25.8% to 36.2%]. Median duration of response was 47.5 months (range, 2.1+ to 51.1+ months; '+' indicates no progressive disease by the time of last disease assessment). Median progression-free survival was 3.5 months (95% CI 2.3-4.2 months) and median overall survival was 20.1 months (95% CI 14.1-27.1 months). Treatment-related adverse events (AEs) occurred in 227 patients (64.7%). Grade 3-4 treatment-related AEs occurred in 39 patients (11.1%); 3 (0.9%) had grade 5 treatment-related AEs (myocarditis, pneumonia, and Guillain-Barre syndrome, n = 1 each). CONCLUSIONS: Pembrolizumab demonstrated clinically meaningful and durable benefit, with a high ORR of 30.8%, long median duration of response of 47.5 months, and manageable safety across a range of heavily pretreated, advanced MSI-H/dMMR noncolorectal cancers, providing support for use of pembrolizumab in this setting.

Health-related quality of life with pembrolizumab monotherapy in patients with previously treated advanced microsatellite instability high/mismatch repair deficient endometrial cancer in the KEYNOTE-158 study.

OBJECTIVE: Pembrolizumab demonstrated a clinically meaningful objective response rate in patients with previously treated, advanced MSI-H/dMMR endometrial cancer in the multicohort phase 2 KEYNOTE-158 study (ClinicalTrials.gov, NCT02628067). We present health-related quality of life (HRQoL) results for these patients. METHODS: This analysis included patients from cohorts D (endometrial cancer with any MSI status) and K (any MSI-H/dMMR solid tumor except colorectal) who had previously treated, advanced MSI-H/dMMR endometrial cancer. Patients received pembrolizumab 200 mg Q3W for 35 cycles. EORTC QLQ-C30 and EQ-5D-3L questionnaires were administered at baseline, at regular intervals during treatment, and 30 days after treatment discontinuation. Pre-specified exploratory analyses included changes from baseline to week 9 in QLQ-C30 global health status (GHS)/QoL and EQ-5D-3L visual analog scale (VAS) score for all patients and by best overall response. RESULTS: 84 of 90 enrolled patients completed ≥1 HRQoL questionnaire and were included in the analysis. QLQ-C30 and EQ-5D-3L compliance rates were 90% and 94%, respectively, at baseline, and 92% and 93% at week 9. Mean (95% CI) QLQ-C30 GHS/QoL scores improved from baseline to week 9 by 6.08 (0.71-11.46) points in the overall population, with greater improvement in patients who achieved complete or partial response (11.67 [5.33-18.00]-point increase). Mean (95% CI) EQ-5D-3L VAS scores improved by 6.00 (2.25-9.75) points in the overall population and 9.11 (5.24-12.98) points in patients with CR/PR. CONCLUSIONS: Pembrolizumab maintained or improved HRQoL in patients with previously treated, advanced MSI-H/dMMR endometrial cancer, further supporting efficacy and safety results from KEYNOTE-158 and pembrolizumab use in this setting.

Precision Constraints for Three-Flavor Neutrino Oscillations from the Full MINOS+ and MINOS Dataset.

We report the final measurement of the neutrino oscillation parameters Δm_{32}^{2} and sin^{2}θ_{23} using all data from the MINOS and MINOS+ experiments. These data were collected using a total exposure of 23.76×10^{20} protons on target producing ν_{µ} and ν[over ¯]_{µ} beams and 60.75 kt yr exposure to atmospheric neutrinos. The measurement of the disappearance of ν_{µ} and the appearance of ν_{e} events between the Near and Far detectors yields |Δm_{32}^{2}|=2.40_{-0.09}^{+0.08}(2.45_{-0.08}^{+0.07})×10^{-3} eV^{2} and sin^{2}θ_{23}=0.43_{-0.04}^{+0.20}(0.42_{-0.03}^{+0.07}) at 68% C.L. for normal (inverted) hierarchy.

Phase Ib study of atezolizumab combined with cobimetinib in patients with solid tumors.

BACKGROUND: Preclinical evidence suggests that MEK inhibition promotes accumulation and survival of intratumoral tumor-specific T cells and can synergize with immune checkpoint inhibition. We investigated the safety and clinical activity of combining a MEK inhibitor, cobimetinib, and a programmed cell death 1 ligand 1 (PD-L1) inhibitor, atezolizumab, in patients with solid tumors. PATIENTS AND METHODS: This phase I/Ib study treated PD-L1/PD-1-naive patients with solid tumors in a dose-escalation stage and then in multiple, indication-specific dose-expansion cohorts. In most patients, cobimetinib was dosed once daily orally for 21 days on, 7 days off. Atezolizumab was dosed at 800 mg intravenously every 2 weeks. The primary objectives were safety and tolerability. Secondary end points included objective response rate, progression-free survival, and overall survival. RESULTS: Between 27 December 2013 and 9 May 2016, 152 patients were enrolled. As of 4 September 2017, 150 patients received ≥1 dose of atezolizumab, including 14 in the dose-escalation cohorts and 136 in the dose-expansion cohorts. Patients had metastatic colorectal cancer (mCRC; n = 84), melanoma (n = 22), non-small-cell lung cancer (NSCLC; n = 28), and other solid tumors (n = 16). The most common all-grade treatment-related adverse events (AEs) were diarrhea (67%), rash (48%), and fatigue (40%), similar to those with single-agent cobimetinib and atezolizumab. One (<1%) treatment-related grade 5 AE occurred (sepsis). Forty-five (30%) and 23 patients (15%) had AEs that led to discontinuation of cobimetinib and atezolizumab, respectively. Confirmed responses were observed in 7 of 84 patients (8%) with mCRC (6 responders were microsatellite low/stable, 1 was microsatellite instable), 9 of 22 patients (41%) with melanoma, and 5 of 28 patients (18%) with NSCLC. Clinical activity was independent of KRAS/BRAF status across diseases. CONCLUSIONS: Atezolizumab plus cobimetinib had manageable safety and clinical activity irrespective of KRAS/BRAF status. Although potential synergistic activity was seen in mCRC, this was not confirmed in a subsequent phase III study. CLINICALTRIALS.GOV IDENTIFIER: NCT01988896 (the investigators in the NCT01988896 study are listed in the supplementary Appendix, available at Annals of Oncology online).

Challenges in initiating and conducting personalized cancer therapy trials: perspectives from WINTHER, a Worldwide Innovative Network (WIN) Consortium trial.

Advances in 'omics' technology and targeted therapeutic molecules are together driving the incorporation of molecular-based diagnostics into the care of patients with cancer. There is an urgent need to assess the efficacy of therapy determined by molecular matching of patients with particular targeted therapies. WINTHER is a clinical trial that uses cutting edge genomic and transcriptomic assays to guide treatment decisions. Through the lens of this ambitious multinational trial (five countries, six sites) coordinated by the Worldwide Innovative Networking Consortium for personalized cancer therapy, we discovered key challenges in initiation and conduct of a prospective, omically driven study. To date, the time from study concept to activation has varied between 19 months at Gustave Roussy Cancer Campus in France to 30 months at the Segal Cancer Center, McGill University (Canada). It took 3+ years to be able to activate US sites due to national regulatory hurdles. Access to medications proposed by the molecular analysis remains a major challenge, since their availability through active clinical trials is highly variable over time within sites and across the network. Rules regarding the off-label use of drugs, or drugs not yet approved at all in some countries, pose a further challenge, and many biopharmaceutical companies lack a simple internal mechanism to supply the drugs even if they wish to do so. These various obstacles should be addressed to test and then implement precision medicine in cancer.

A phase I dose escalation trial of tremelimumab (CP-675,206) in combination with gemcitabine in chemotherapy-naive patients with metastatic pancreatic cancer.

BACKGROUND: Tremelimumab (CP-675,206) is a fully human monoclonal antibody binding to cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) on T cells that stimulates the immune system by blocking the CTLA4-negative regulatory signal. Combination with standard chemotherapy may strengthen antitumor therapy. This is a phase Ib, multisite, open-label, nonrandomized dose escalation trial evaluating the safety, tolerability, and maximum tolerated dose (MTD) of tremelimumab combined with gemcitabine in patients with metastatic pancreatic cancer. PATIENTS AND METHODS: Gemcitabine (1000 mg/m(2) on days 1, 8, and 15 of each 28-day cycles) was administrated with escalating doses of i.v. tremelimumab (6, 10, or 15 mg/kg) on day 1 of each 84-day cycle for a maximum of 4 cycles. The first 18 patients had an initial 4-week gemcitabine-only lead-in period. Dose-limiting toxicities (DLTs) related to tremelimumab were evaluated during the first 6 weeks after the first dose of tremelimumab. RESULTS: From June 2008 to August 2011, 34 patients were enrolled and received at least one dose of tremelimumab. No DLTs related to tremelimumab were observed at any dose, even when the maximum dose established for tremelimumab (15 mg/kg) was used. Most frequent grade 3/4 toxicities were asthenia (11.8%) and nausea (8.8%). Only one patient had a serious drug-related event (diarrhea with dehydration). The median overall survival was 7.4 months (95% confidence interval 5.8-9.4 months). At the end of treatment, two patients achieved partial response. Both patients received tremelimumab 15-mg/kg group (n = 2/19, 10.5%). CONCLUSION: Tremelimumab plus gemcitabine demonstrated a safety and tolerability profile, warranting further study in patients with metastatic pancreatic cancer. CLINICALTRIALSGOV ID: NCT00556023.

Patient perception of the benefit of a BRAF inhibitor in metastatic melanoma: quality-of-life analyses of the BREAK-3 study comparing dabrafenib with dacarbazine.

BACKGROUND: In a randomized phase III study (BREAK-3), dabrafenib showed prolonged progression-free survival (PFS) (median 5.1 versus 2.7 months; hazard ratio = 0.30; 95% confidence interval 0.18-0.53; P < 0.0001) compared with dacarbazine (DTIC) in patients with BRAF V600E metastatic melanoma. Assessing how these results are transformed into a real health benefit for patients is crucial. METHODS: The EORTC QLQ-C30 questionnaire assessed quality of life (QoL) at baseline and follow-up visits. RESULTS: For DTIC, all functional dimensions except role dimension worsened from baseline at follow-up. For dabrafenib, all functionality dimensions remained stable relative to baseline or improved at week 6; mean change in seven symptom dimensions improved from baseline, with appetite loss, insomnia, nausea and vomiting, and pain showing the greatest improvement. In the DTIC arm, symptom dimensions were unchanged or worsened from baseline for all symptoms except pain (week 6), with the greatest exacerbations observed for fatigue and nausea and vomiting. Mixed-model-repeated measures analyses showed significant (P < 0.05) and/or clinically meaningful improvements from baseline in favor of dabrafenib for emotional and social functioning, nausea and vomiting, appetite loss, diarrhea, fatigue, dyspnea, and insomnia at weeks 6 and/or 12. After crossing over to dabrafenib upon progression (n = 35), improvements in all QoL dimensions were evident after receiving dabrafenib for 6 (n = 31) to 12 (n = 25) weeks. CONCLUSIONS: This first reported QoL analysis for a BRAF inhibitor in metastatic melanoma demonstrates that the high tumor response rates and PFS superiority of dabrafenib over DTIC is not only a theoretical advantage, but also transforms in a rapid functional and symptomatic benefit for the patient. ClinicalTrials.gov Identifier: NCT01227889.

Ki 67 is a major, but not the sole determinant of Oncotype Dx recurrence score.

BACKGROUND: Immunohistological assessment of Ki 67 expression is less expensive than Oncotype Dx, which is currently used to identify patients with lymph node-negative breast cancer, who will benefit from adjuvant chemotherapy. METHODS: The relationship of immunohistologically measured Ki 67 to Oncotype DX recurrence score (RS) was examined in 53 cases of T1-2 N0 M0 (oestrogen receptor-positive, HER2/neu negative) breast cancer. RESULTS: There was a strong linear correlation between Ki 67 value and the Oncotype Dx RS. All patients in the low Ki 67 group (Ki 67 of ≤ 10%) had Oncotype Dx RSs of low or intermediate risk. The vast majority of patients (93.8%) in the high-Ki 67 group (Ki 67 ≥ 25%) had oncotype RSs of high or intermediate risk. CONCLUSION: Ki 67 proliferation value is a major, but not the sole determinant of Oncotype Dx score.

A review of cancer immunotherapy: from the past, to the present, to the future.

Compared with previous standards of care (including chemotherapy, radiotherapy, and surgery), cancer immunotherapy has brought significant improvements for patients in terms of survival and quality of life. Immunotherapy has now firmly established itself as a novel pillar of cancer care, from the metastatic stage to the adjuvant and neoadjuvant settings in numerous cancer types. In this review article, we highlight how the history of cancer immunotherapy paved the way for discoveries that are now part of the standard of care. We also highlight the current pitfalls and limitations of cancer checkpoint immunotherapy and how novel research in the fields of personalized cancer vaccines, autoimmunity, the microbiome, the tumour microenvironment, and metabolomics is aiming to solve those challenges.

Semiclassical methods in chemical physics.

Semiclassical theory finds use in chemical physics both as a computational method and as a conceptual framework for interpreting quantum features in experiments and in numerical quantum calculations. The semiclassical description of one-dimensional dynamical systems is essentially a solved problem for eigenvalue and scattering situations and for general topologies of potential functions (simple potential wells, multiple wells, multiple barriers, and so forth). Considerable progress has also been made in generalizing semiclassical theory to multidimensional dynamical systems (such as inelastic and reactive scattering of atoms and molecules and vibrational energy levels of polyatomic molecules), and here, too, it provides a useful picture of quantum features (interference in product state distribution, generalized tunneling phenomena, and others) in these more complex systems.

Cyclic adenosine monophosphate: function in photoreceptors.

Inactivation of adenylate cyclase in outer segments of retinal photoreceptor cells is proportional to the bleaching of rhodopsin. Membranes of the outer segments also contain a particulate, light-insensitive phosphodiesterase of high specific activity. In electrophysiological experiments, application of cyclic adenosine monophosphate along with a methylxanthine mimics the effects of illumination on the photoreceptor cell of the compound eye of Limulus.

Digitonin effects on photoreceptor adenylate cyclase.

Adenylate cyclase is described in a number of photoreceptor membranes. Vertebrate rod outer segments contain light-regulated cyclase, and light regulation is abolished by digitonin. Disruption of microvilli in cone and rhabdomphotoreceptors is also associated with loss of light regulation and retention of full enzymic activity. The data suggest that inhibitory constraint provides regulation in cyclase systems and that disruption of membrane structure uncouples catalytic and regulatory elements.

Phase I clinical trial of i.v. ascorbic acid in advanced malignancy.

BACKGROUND: Ascorbic acid is a widely used and controversial alternative cancer treatment. In millimolar concentrations, it is selectively cytotoxic to many cancer cell lines and has in vivo anticancer activity when administered alone or together with other agents. We carried out a dose-finding phase I and pharmacokinetic study of i.v. ascorbic acid in patients with advanced malignancies. PATIENTS AND METHODS: Patients with advanced cancer or hematologic malignancy were assigned to sequential cohorts infused with 0.4, 0.6, 0.9 and 1.5 g ascorbic acid/kg body weight three times weekly. RESULTS: Adverse events and toxicity were minimal at all dose levels. No patient had an objective anticancer response. CONCLUSIONS: High-dose i.v. ascorbic acid was well tolerated but failed to demonstrate anticancer activity when administered to patients with previously treated advanced malignancies. The promise of this approach may lie in combination with cytotoxic or other redox-active molecules.

Trolox enhances the anti-lymphoma effects of arsenic trioxide, while protecting against liver toxicity.

Arsenic trioxide (As2O3) is an effective therapy in acute promyelocytic leukemia (APL), but its use in other malignancies is limited by the higher concentrations required to induce apoptosis. We have reported that trolox, an analogue of alpha-tocopherol, increases As2O3-mediated apoptosis in a variety of APL, myeloma and breast cancer cell lines, while non-malignant cells may be protected. In the present study, we extended previous results to show that trolox increases As2O3-mediated apoptosis in the P388 lymphoma cell line in vitro, as evidenced by decrease of mitochondrial membrane potential and release of cytochrome c. We then sought to determine whether this combination can enhance antitumor effects while protecting normal cells in vivo. In BDF1 mice, trolox treatment decreased As2O3-induced hepatomegaly, markers of oxidative stress and hepatocellular damage. In P388 tumor-bearing mice, As2O3 treatment prolonged survival, and the addition of trolox provided a further significant increase in lifespan. In addition, the combination of As2O3 and trolox inhibited metastatic spread, and protected the tumor-bearing mice from As2O3 liver toxicity. Our results suggest, for the first time, that trolox might prevent some of the clinical manifestations of As2O3-related toxicity while increasing its pro-apoptotic capacity and clinical efficacy in hematological malignancies.

Systematic review with meta-analysis: the worldwide prevalence of Helicobacter pylori infection.

BACKGROUND: The epidemiology of Helicobacter pylori infection is poorly understood. AIM: To establish the reported regional and national prevalence of H. pylori infection, stratified by age and gender. METHODS: All relevant English publications from 2000 to 2017 cited by PubMed and Scopus were retrieved using comprehensive combinations of keywords. The overall prevalence of H. pylori was estimated using both random effect and fixed effect meta-analyses, and presented as prevalence rate (% and 95% CI). The analyses were extended by separation into gender and age groups. RESULTS: A total of 14 056 records were obtained initially. After applying exclusion criteria in several steps, 183 studies were selected. Analysis of 410 879 participants from 73 countries in six continents revealed an overall prevalence of 44.3% (95% CI: 40.9-47.7) worldwide. This rate ranged from 50.8% (95% CI: 46.8-54.7) in developing countries compared with 34.7% (95% CI: 30.2-39.3) in developed countries. The global H. pylori infection rate was 42.7% (95% CI: 39-46.5) in females compared to 46.3% (95% CI: 42.1-50.5) in males. The prevalence in adults (≥18 years) was significantly higher than in children (48.6% [95% CI: 43.8-53.5] vs 32.6% [95% CI: 28.4-36.8], respectively). There was a statistically nonsignificant decrease in the prevalence in 2009-2016 compared with the 2000-2009 period. CONCLUSIONS: The observed differences between countries appear to be due to economic and social conditions. H. pylori infection can be a benchmark for the socioeconomic and health status of a country. Further studies are suggested to investigate the natural history of the acquisition of H. pylori infection from childhood into adult life.

CCAAT/enhancer binding protein epsilon is a potential retinoid target gene in acute promyelocytic leukemia treatment.

The CCAAT/enhancer binding protein epsilon (C/EBPepsilon) is a nuclear transcription factor expressed predominantly in myeloid cells and implicated as a potential regulator of myeloid differentiation. We show that it was rapidly induced in the acute promyelocytic leukemia (APL) cell line NB4 during granulocytic differentiation after exposure to retinoic acid (RA). Our data suggest that induction of C/EBPepsilon expression was through the retinoic acid receptor alpha (RARalpha) pathway. Reporter gene studies showed that C/EBPepsilon promoter/enhancer activity increased in a retinoid-dependent fashion via the retinoic acid response element (RARE) present in the promoter region of C/EBPepsilon. The RA-induced expression of C/EBPepsilon markedly increased in U937 myelomonoblasts that were induced to express promyelocytic leukemia/RARalpha (PML/RARalpha), but not in those induced to express promyelocytic leukemia zinc finger/RARalpha (PLZF/RARalpha). In retinoid-resistant APL cell lines, C/EBPepsilon either is not induced or is induced only at very high concentrations of RA (>/=10(-6) M). In addition, forced expression of C/EBPepsilon in the U937 myelomonoblastic leukemia cells mimicked terminal granulocytic differentiation, including morphologic changes, increased CD11b/CD66b expression, and induction of secondary granule protein expression. Our data strongly suggest that C/EBPepsilon is a downstream target gene responsible for RA-induced granulocytic differentiation of APL cells.

Antitumor effects of doxorubicin in combination with anti-epidermal growth factor receptor monoclonal antibodies.

BACKGROUND: A variety of human tumors frequently express high levels of epidermal growth factor (EGF) receptor and its ligand, transforming growth factor alpha (TGF-alpha), which in some tumors is associated with poor prognosis. Monoclonal antibodies (MAbs) that block the binding of TGF-alpha or EGF to the receptor can inhibit proliferation of tumor cells that express the receptor. Studies suggest that these MAbs may enhance the antitumor effects of chemotherapy. PURPOSE: Our purpose was to study, in vitro and in vivo, the antitumor effects of doxorubicin in combination with anti-EGF receptor MAbs against tumor cells expressing high levels of EGF receptor. Our goal was to achieve maximum initial cytoreduction with high-dose doxorubicin in association with prolonged blockade of EGF receptor with MAbs. METHODS: Anti-EGF receptor MAbs 528 (isotype IgG2a) and 225 (isotype IgG1) were used in combination with doxorubicin against cells from human A431 squamous cell carcinoma and human MDA-468 breast adenocarcinoma. Both A431 and MDA-468 cells express high levels of EGF receptors and TGF-alpha. Cultured cells were treated with doxorubicin (range, 0-10 nM) in the presence or absence of MAb 528 or 225 (range, 0-30 nM). At 48 hours, doxorubicin-containing medium was removed, and treatment with antibody was continued for 5 days, when cell proliferation assays were performed. The activity of the agents and the combinations against well-established xenografts in BALB/c nude mice was also studied. In nude mice, doxorubicin was given at doses of 50-100 micrograms/20 g body weight on 2 successive days, and MAbs 528 and 225 were given at a dose range of 0-2 mg intraperitoneally twice a week. RESULTS: MAbs 528 and 225 both enhanced the antitumor effects of doxorubicin against A431 and MDA-468 tumor cells, producing additive growth suppression in cell cultures. MAb 528 increased the antitumor effects of doxorubicin by 32%-42%, and similar results were obtained with MAb 225. In BALB/c athymic mice, the treatment of well-established xenografts with either doxorubicin or anti-EGF receptor MAb alone temporarily inhibited growth, but the combination of both agents substantially enhanced antitumor activity over that of doxorubicin alone in A431 and MDA-468 cell xenografts. The combination treatment of mice bearing A431 xenografts resulted in tumor eradication of 40%-100% in the surviving mice in several independent experiments. The enhanced antitumor activity was dose dependent. CONCLUSIONS: Our results suggest that anti-EGF receptor MAbs substantially enhance the effects of doxorubicin against well-established xenografts of tumor cells expressing high levels of EGF receptors. IMPLICATIONS: Clinical trials with anti-EGF receptor MAbs are being conducted, and trials with anti-EGF receptor MAbs combined with doxorubicin are planned.

Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR alpha protein in acute promyelocytic leukemia cells.

BACKGROUND: Retinoids, which are derivatives of vitamin A, induce differentiation of acute promyelocytic leukemia (APL) cells in vitro and in patients. However, APL cells develop resistance to retinoic acid treatment. Arsenic trioxide (As2O3) can induce clinical remission in patients with APL, including those who have relapsed after retinoic acid treatment, by inducing apoptosis (programmed cell death) of the leukemia cells. In this study, we investigated the molecular mechanisms by which As2O3 induces apoptosis in retinoic acid-sensitive NB4 APL cells, in retinoic acid-resistant derivatives of these cells, and in fresh leukemia cells from patients. METHODS: Apoptosis was assessed by means of DNA fragmentation analyses, TUNEL assays (i.e., deoxyuridine triphosphate labeling of DNA nicks with terminal deoxynucleotidyl transferase), and flow cytometry. Expression of the PML/RAR alpha fusion protein in leukemia cells was assessed by means of western blotting, ligand binding, and immunohistochemistry. Northern blotting and ribonuclease protection assays were used to evaluate changes in gene expression in response to retinoic acid and As2O3 treatment. RESULTS AND CONCLUSIONS: As2O3 induces apoptosis without differentiation in retinoic acid-sensitive and retinoic acid-resistant APL cells at concentrations that are achievable in patients. As2O3 induces loss of the PML/RAR alpha fusion protein in NB4 cells, in retinoic-acid resistant cells derived from them, in fresh APL cells from patients, and in non-APL cells transfected to express this protein. As2O3 and retinoic acid induce different patterns of gene regulation, and they inhibit the phenotypes induced by each other. Understanding the molecular basis of these differences in the effects of As2O3 and retinoic acid may guide the clinical use of arsenic compounds and provide insights into the management of leukemias that do not respond to retinoic acid.

A ribozyme which discriminates in vitro between PML/RAR alpha, the t(15;17)-associated fusion RNA of acute promyelocytic leukemia, and PML and RAR alpha, the transcripts from the nonrearranged alleles.

Acute promyelocytic leukemia (FAB M3) is distinguished by the presence of the t(15;17) and clinical response to all-trans retinoic acid (RA) treatment. Acute promyelocytic leukemia is associated with a chromosomal translocation which results in the fusion of genes encoding a putative transcription factor (PML) and the retinoic acid receptor alpha (RAR alpha). It is suggested that the PML/RAR alpha fusion protein functions as an inhibitor of myeloid differentiation. The potential use of ribozymes as therapeutic agents has been investigated in the present study. Hammerhead ribozymes, which by hybridizing to both PML and RAR alpha sequences discriminate between the fusion transcript and the normal transcripts from the nonrearranged alleles, were designed and synthesized. Two hammerhead cleavage sites were targeted: site 1, an AUU located 4 nucleotides 3' to the fusion junction; and site 2, a UUC located 26 nucleotides 3' to the junction. Both sites are located in the RAR alpha portion of the fusion transcript. Using a full-length PML/RAR alpha RNA or an RNA corresponding to 788 nucleotides of the PML/RAR alpha mRNA and a full-length RAR alpha RNA or an RNA corresponding to 960 nucleotides of the RAR alpha mRNA as model substrates, the catalytic behavior of several ribozymes was studied. A modified hammerhead directed against site 2 displayed the highest degree of selectivity for PML/RAR alpha. It is hypothesized that ribozyme-mediated inactivation of PML/RAR alpha provides a new approach to study the role of PML/RAR alpha in the deregulated growth and RA response of acute promyelocytic leukemia.

Estrogen receptor expression activates the transcriptional and growth-inhibitory response to retinoids without enhanced retinoic acid receptor alpha expression.

Estrogen receptor (ER)-positive human breast cancer cells are hormonally regulated and are inhibited by retinoids, whereas most ER-negative breast cancer cells are not. Here, we compared retinoid-induced transcriptional activation and growth inhibition in the ER-negative breast cancer cell line MDA-MB-231, stably transfected to express wild-type ER (S30), with that of the ER-positive MCF-7 line and the ER-negative parental line. Retinoids inhibited growth of the ER-expressing S30 clone but not of the parental MDA-MB-231 cells. Unlike a previously reported MDA-MD-231 subclone that was transfected to express a mutated ER (G400V), S30 did not express increased levels of retinoid receptor RNA or protein, nor was there increased binding activity to retinoid-responsive DNA elements. However, stable expression of ER increased retinoid activation of transcription of a retinoic acid (RA) response elements from the low level in MDA-MB-231 to approach the level of MCF-7. The restored growth inhibition and transcriptional regulation by RA were unaffected by treatment with ER agonists or antagonists. Transient expression of ER but not of other nuclear receptors in MDA-MB-231 cells also activated retinoid-induced transcription, showing that this response is specific to ER. Furthermore, the effect of exogenously expressed ER on retinoid response was much greater than that obtained by overexpression of RA receptor alpha and/or retinoid X receptor alpha. Finally, a panel of ER mutants showed that enhancement of retinoid-induced transcriptional activity was dependent on the integrity of the DNA binding domain.