Molecular patterns of response and treatment failure after frontline venetoclax combinations in older patients with AML.

The BCL-2 inhibitor venetoclax combined with hypomethylating agents or low-dose cytarabine represents an important new therapy for older or unfit patients with acute myeloid leukemia (AML). We analyzed 81 patients receiving these venetoclax-based combinations to identify molecular correlates of durable remission, response followed by relapse (adaptive resistance), or refractory disease (primary resistance). High response rates and durable remissions were typically associated with NPM1 or IDH2 mutations, with prolonged molecular remissions prevalent for NPM1 mutations. Primary and adaptive resistance to venetoclax-based combinations was most commonly characterized by acquisition or enrichment of clones activating signaling pathways such as FLT3 or RAS or biallelically perturbing TP53. Single-cell studies highlighted the polyclonal nature of intratumoral resistance mechanisms in some cases. Among cases that were primary refractory, we identified heterogeneous and sometimes divergent interval changes in leukemic clones within a single cycle of therapy, highlighting the dynamic and rapid occurrence of therapeutic selection in AML. In functional studies, FLT3 internal tandem duplication gain or TP53 loss conferred cross-resistance to both venetoclax and cytotoxic-based therapies. Collectively, we highlight molecular determinants of outcome with clinical relevance to patients with AML receiving venetoclax-based combination therapies.

Targeting antigen-independent proliferation in chronic lymphocytic leukemia through differential kinase inhibition.

The clinical success of B-cell receptor (BCR) signaling pathway inhibitors in chronic lymphocytic leukemia (CLL) is attributed to inhibition of adhesion in and migration towards the lymph node. Proliferation of CLL cells is restricted to this protective niche, but the underlying mechanism(s) is/are not known. Treatment with BCR pathway inhibitors results in rapid reductions of total clone size, while CLL cell survival is not affected, which points towards inhibition of proliferation. In vitro, BCR stimulation does not induce proliferation of CLL, but triggering via Toll-like receptor, tumor necrosis factor or cytokine receptors does. Here, we investigated the effects of clinically applied inhibitors that target BCR signaling, in the context of proliferation triggered either via CD40L/IL-21 or after CpG stimulation. CD40L/IL-21-induced proliferation could be inhibited by idelalisib and ibrutinib. We demonstrate this was due to blockade of CD40L-induced ERK-signaling. Targeting JAKs, but not SYK, blocked CD40L/IL-21-induced proliferation. In contrast, PI3K, BTK as well as SYK inhibition prevented CpG-induced proliferation. Knockdown experiments showed that CD40L/IL-21 did not co-opt upstream BCR components such as CD79A, in contrast to CpG-induced proliferation. Our data indicate that currently applied BTK/PI3K inhibitors target antigen-independent proliferation in CLL, and suggest that targeting of JAK and/or SYK might be clinically useful.

The pan phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) blocks survival, adhesion and proliferation of primary chronic lymphocytic leukemia cells.

The phosphoinositide 3-kinases (PI3Ks) are critical components of the B-cell receptor (BCR) pathway and have an important role in the pathobiology of chronic lymphocytic leukemia (CLL). Inhibitors of PI3Kδ block BCR-mediated cross-talk between CLL cells and the lymph node microenvironment and provide significant clinical benefit to CLL patients. However, the PI3Kδ inhibitors applied thus far have limited direct impact on leukemia cell survival and thus are unlikely to eradicate the disease. The use of inhibitors of multiple isoforms of PI3K might lead to deeper remissions. Here we demonstrate that the pan-PI3K/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) was more pro-apoptotic to CLL cells--irrespective of their ATM/p53 status--than PI3Kα or PI3Kδ isoform selective inhibitors. Furthermore, SAR245409 blocked CLL survival, adhesion and proliferation. Moreover, SAR245409 was a more potent inhibitor of T-cell-mediated production of cytokines, which support CLL survival. Taken together, our in vitro data provide a rationale for the evaluation of a pan-PI3K inhibitor in CLL patients.

CLL cells are resistant to smac mimetics because of an inability to form a ripoptosome complex.

In the lymph node (LN) environment, chronic lymphocytic leukemia (CLL) cells display increased NF-κB activity compared with peripheral blood CLL cells, which contributes to chemoresistance. Antagonists of cellular inhibitor of apoptosis proteins (cIAPs) can induce apoptosis in various cancer cells in a tumor necrosis factor-α (TNFα)-dependent manner and are in preclinical development. Smac-mimetics promote degradation of cIAP1 and cIAP2, which results in TNFR-mediated apoptosis via formation of a ripoptosome complex, comprising RIPK1, Fas-associated protein with death domain, FLICE-like inhibitory protein and caspase-8. CD40 stimulation of CLL cells in vitro is used as a model to mimic the LN microenvironment and results in NF-κB activation and TNFα production. In this study, we investigated the response of CLL cells to smac-mimetics in the context of CD40 stimulation. We found that treatment with smac-mimetics results in cIAP1 and cIAP2 degradation, yet although TNFα is produced, this did not induce apoptosis. Despite the presence of all components, the ripoptosome complex did not form upon smac-mimetic treatment in CLL cells. Thus, CLL cells seem to possess aberrant upstream NF-κB regulation that prevents ripoptosome formation upon IAP degradation. Unraveling the exact molecular mechanisms of disturbed ripoptosome formation may offer novel targets for treatment in CLL.

Radiofrequency induced endometrial ablation: an update.

OBJECTIVE: To test the safety and efficiency of radiofrequency endometrial ablation as a nonhysteroscopic treatment of dysfunctional uterine bleeding. DESIGN: A multicentre trial. SETTING: Nineteen clinics in six countries. METHODS: From February 1990 to December 1994, 1280 women were treated with radiofrequency endometrial ablation. Inclusion criteria were: menorrhagia, age 30-55 years, a completed family, a wish to retain the uterus, no hypergonadotropic state indicating an approaching menopause, a normal sized uterus, normal cervical cytology, normal adnexa, no prolapse, no intrauterine abnormalities, and no history of a bleeding disorder. Treatment was performed according to a standard operating protocol. RESULTS: Either amenorrhoea or a satisfactory improvement of menstruation was obtained in 78.5% of 944 women followed for six months or more. The design of the equipment has been thoroughly revised and improved during the last four years. The complications encountered were mostly related to the handling of radiofrequency and sometimes due to failures in following the safety protocol. CONCLUSIONS: Although the technology is complicated, the treatment is simple, fast and effective. The complications have often been unpredictable. Despite the improvements made during this period, safety must be further enhanced to develop the original concept into an established technique.

[Non-hysteroscopic thermodestruction of the endometrium using radio waves in the treatment of menorrhagia]. / Niet-hysteroscopische thermodestructie van het endometrium door radiogolven ter behandeling van menorragie.

OBJECTIVE: To test non-hysteroscopic thermodestruction of the endometrium for safety and efficiency. DESIGN: Prospective pilot study from February 1991 to July 1992. SETTING: University Hospital Nijmegen. PATIENTS AND METHOD: There were 68 patients aged 30-55 years with menorrhagia, who did not want children but wished to retain the uterus. There were two patients with a subserous myoma but with a normal uterine cavity. Patients with hypergonadotropism, a markedly enlarged uterus, abnormalities at cytological examination of the cervix, adnexal lesions, prolapse, intrauterine lesions and coagulation disorders were excluded. By way of preparation, danazol was administered for 4 weeks before the intervention (36 patients). Three patients with side effects were given lynestrenol. From January 1992, an LH-RH analogue was administered (29 patients) for 6 weeks prior to the intervention or in the patients with a subserous myoma for 12 weeks. Thermodestruction of the endometrium by means of radio waves was carried out according to a standard protocol under general or epidural spinal anaesthesia. The intracavitary temperature measured was 62-65 degrees C, mean duration of the treatment 20 minutes. RESULTS: The patients were discharged 10-24 hours after the intervention. A non-disturbing watery discharge of 3-6 weeks' duration was reported. Normal activities were resumed after one week. Success (duration of follow-up 3-21 months) was defined as amenorrhoea (6 patients; 9%) or a markedly reduced menstruation or duration of menstruation (48 patients; 70%). Six patients (9%) reported no improvement and of eight patients (12%) with slight, unsatisfactory improvement three were treated again, with success (4%). During the trial period the method was further adjusted and perfected. There were no complications. CONCLUSION: This preliminary experience shows that thermodestruction with radio waves is simple, safe and efficient.

Avian herpesvirus as a live viral vector for the expression of heterologous antigens.

Control of Marek's disease in the poultry industry has been successfully achieved for several decades by large-scale vaccination of day-old chickens with live herpesvirus of turkeys (HVT) strains. Several features of this virus including lack of pathogenicity and long-term immune protection due to a persistent viraemic infection made us decide to use HVT as a live viral vector for the expression of foreign antigens. Potential sites for the integration of foreign DNA in the unique short region of the HVT genome were identified by the insertion of a beta-galactosidase expression cassette. Vaccination trials with recombinant virus strains indicated that the marker gene was expressed and stably maintained during animal passage. Based on an insertion site mapping in one of the open reading frames of the unique short region, a general recombination vector was designed for the integration of foreign genes into HVT. Recombinant virus-directed expression of individual antigens from Newcastle disease virus was driven by a strong promoter element derived from the lung terminal repeat sequence of Rous sarcoma virus.

Successful pregnancy after ZIFT in a patient with congenital cervical atresia.

We report the third patient with a successful pregnancy following operative correction of congenital cervical atresia. The pregnancy was achieved through zygote intrafallopian transfer (ZIFT). Although stenosis of the newly formed cervical canal causes considerable morbidity, therapy should be aimed at the creation of a conduit for menstrual blood and for possible future pregnancy. New techniques in assisted reproduction such as in vitro fertilization-embryo transfer, gamete intrafallopian transfer, and ZIFT increase the chances of pregnancy in patients with congenital cervical atresia. A hysterectomy, as advocated until very recently, should not, in our opinion, be the first treatment of choice.

Quantitation of conjugate formation between human polymorphonuclear leukocytes and antibody-coated target cells by flow cytometry: the role of Fc receptor and LFA-1 antigen.

The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells was characterized by flow cytometry. PMN were labeled with phycoerythrin-E (PE) via a granulocyte-specific monoclonal antibody (leu-M1) and mixed with fluorescein isothiocyanate-labeled K562 tumor cells sensitized with rabbit antiserum. Specific conjugates were formed as analyzed by two-color fluorescence in a flow cytometer. The formation of stable conjugates was dependent on initiation of contact, temperature, time, and antiserum concentration. Studies with inhibitors implicate that microfilaments, but not microtubules, Ca2+, Mg2+, or energy-dependent processes were a prerequisite for binding of PMN to the antibody-coated target cells. No conjugates were formed when uncoated target cells were used or when the experiment was performed in the presence of protein A, indicating that binding was specifically mediated through Fc receptors (FcR). Monoclonal antibodies against the FcRII and FcRIII were used to address the role of these receptors in conjugation. One of the two anti-FcRIII antibodies and an anti-FcRII antibody effectively prevented conjugation. A monoclonal antibody directed against the common beta-chain of the adhesion molecule family and a combination of antibodies against the alpha-chain of LFA-1 and Mo-1 also blocked conjugation when target cells were sensitized under suboptimal conditions. The antibody against the beta-chain also diminished killing of antibody-coated K562, as measured by chromium release when included in the cytotoxicity assay. These results indicate that flow cytometry permits accurate quantitation and characterization of the binding between PMN and antibody-coated target cells, which in principle, can be prevented by monoclonal antibodies against surface receptors. Binding is primarily established by both the FcRII and FcRIII. Adhesion-associated molecules on the PMN surface contribute to optimal binding.

Is there influence of smoking on the mutagenicity of follicular fluid?

The mutagenicity of follicular fluid was examined in 24 patients, 12 smoking and 12 nonsmoking, who were treated in an in vitro fertilization program. The Salmonella microsome assay was used. It was found that the mutagenicity of follicular fluid was not influenced by the number of cigarettes smoked. Urine samples of smoking in vitro fertilization (IVF) patients however showed a dose-dependent elevation of the mutagenicity.

Eating, drinking, and cycling. A controlled Tour de France simulation study, Part I.

Sustained exhausting exercise is thought to depress appetite and food intake. The aim of the present investigation was to study the effect of intensive cycling exercise, with an energy expenditure comparable to values derived from the Tour de France, on food and fluid intake, energy balance, nitrogen balance, and nutrient oxidation. Thirteen highly trained cyclists consuming a normal carbohydrate (CHO)-rich diet (60 En%) were studied during a 7-day stay in a respiration chamber. Two preparation days were followed by a standardized resting day (3), after which the subjects completed two exhausting exercise days (4-5). On day 6 the standardized resting day was repeated. Food and fluid intake were measured by weighed procedure. Energy expenditure was calculated from continuous gas analysis. Energy and nitrogen losses were calculated from all measured excretes. The results showed that energy balance (EB) and nitrogen balance (NB) were positive on the first resting day and became negative on the exercise days. EB was positive again on the recovery day whereas NB remained negative. Nitrogen losses almost balanced N intakes (1.7 g.kg-1) indicating an increased protein requirement. CHO oxidation exceeded CHO intake indicating endogenous CHO depletion. Contribution of CHO to energy exchange decreased from 51.4% +/- 3.1% on day 4 to 40.6% +/- 3.4% on day 5; this decrease was compensated by an increased fat oxidation. The food consumption pattern during days 4 and 5 was not different from days 2 and 6. In-between meal consumption accounted for 30.5%-34.3% of total energy intake. Fluid consumption was adequate to compensate for the losses.(ABSTRACT TRUNCATED AT 250 WORDS)

Eating, drinking, and cycling. A controlled Tour de France simulation study, Part II. Effect of diet manipulation.

Field studies during the Tour de France indicated that cyclists consume 30% of daily energy intake as liquid carbohydrate (CHO)-enriched nutrition with the goal of maintaining energy and CHO balance. The aim of the present investigation was to study the effect of such dietary manipulation during 2 days of long-lasting exhausting cycling on food and fluid intake, energy balance, nitrogen balance, and nutrient oxidation. Thirteen highly trained cyclists were divided into two subgroups receiving ad libitum either a primarily maltodextrin-based beverage (Mf) (20% w/v, 85% maltodextrin, 15% fructose) or a 50/50% composed fructose-maltodextrin (FM) beverage in addition to their normal diet. The study was performed during a 7-day stay in a respiration chamber (2 preparation days, 1 standardized resting day, 2 cycling days, 1.5 standardized recovery days), allowing for continuous gas analysis, weighed food and fluid intake procedure, and collection of excretes. The data of this study were compared with data from the same subjects receiving a normal CHO-rich diet (N) (60 En%) in a separate experiment. The results showed that the cyclists receiving Mf were able to maintain EB during sustained exercise days in contrast to when receiving N and to subjects receiving FM. With Mf treatment CHO intake increased, up to 80 En% (17.5 +/- 1.0 g.kg-1 BW) and carbohydrate balance remained positive. The subjects receiving FM had the largest CHO oxidation, calculated from R. Protein oxidation significantly increased in N and FM as a result of exercise but not in Mf. The latter subjects were in slightly negative nitrogen balance at a protein intake level of 1.4 g.kg-1 BW.(ABSTRACT TRUNCATED AT 250 WORDS)

Serological classification of recent infectious bronchitis virus isolates by the neutralization of immunofluorescent FOCI.

The neutralisation of immunofluorescent foci test was adapted to the grouping of 14 recent Dutch infectious bronchitis virus isolates. This test provides a distinct grouping of the isolates and the corresponding sera. Evaluation of the tests was carried out by means of the computer program called 'Taxonomic', designed for the calculation of taxonomic order from serological data. The taxonomic order, depicted in the form of a tree, facilitates the judgement of the degree of resemblance between viruses or groups of viruses as well as sera. Using this test the isolates of infectious bronchitis virus can be classified into distinct groups.

Environmental and hormonal control of the seasonal egg laying period in field specimens of Lymnaea stagnalis.

A study has been made in field specimens of Lymnaea stagnalis of the relationship between environmental factors (temperature, photoperiod, and food) and spontaneous ovipository activity as well as oviposition evoked by injection of the ovulation hormone (CDCH) during a 1-year cycle. It appeared that spontaneous egg laying started in mid-May and ended in mid-September. It is concluded that it is the quantity of assimilated food that triggers the onset of the egg laying season. Its termination is very likely determined by a synergistic action of environmental factors. It is suggested that these factors control the activities of the CDCH-producing neuro-endocrine caudo-dorsal cells. In winter the snails are completely insensitive to injected CDCH, although many large oocytes are present in the gonad. During 2 months before and after the egg laying season, however, many injected snails respond to CDCH. It is argued that these phenomena are caused by changes in the activities of the endocrine dorsal bodies, which control vitellogenesis and the activities of the female accessory sex organs. The changes in the activities of the dorsal bodies are probably controlled by the synergistic actions of the previously mentioned environmental factors. Oviposition latency (interval between CDCH injection and start of oviposition) is much shorter during the egg laying season than in the nonreproductive period. This must be ascribed to the direct effects of temperature.

Effects of lysosomotropic amines on human polymorphonuclear leucocyte function.

Lysosomotropic agents interfere with lysosome function. We studied the effects of the lysosomotropic amines: lidocaine, diphenylamine and dansylcadaverine on several functions of human polymorphonuclear leucocytes (PMN): enzyme release, phagosome-lysosome fusion, superoxide anion generation upon stimulation with opsonized bacteria, and phagocytosis and killing of opsonized Staphylococcus aureus. Lidocaine depressed all cellular functions tested. Diphenylamine reduced enzyme release and phagosome-lysosome fusion in phagocytosing PMN. This was accompanied by an increase in superoxide anion generation. Dansylcadaverine enhanced enzyme release and phagosome-lysosome fusion, and reduced superoxide anion generation. Neither of these two agents influenced bacterial uptake; bacterial killing was impaired only in dansylcadaverine treated cells. Cadaverine, an analogue that does not penetrate cells, had no effect on any of the functions tested.