A Multicenter Randomized Bioequivalence Study of a Novel Ready-to-Use Temozolomide Oral Suspension vs. Temozolomide Capsules.

BACKGROUND: Temozolomide (TMZ) oral suspension (Ped-TMZ, KIZFIZO®) is being developed for the treatment of relapsed or refractory neuroblastoma, a rare cancer affecting infants and young children. The study assessed the safety and the bioequivalence of this novel pediatric formulation with existing TMZ oral capsules. METHODS: In vitro dissolution profiles and the bioequivalence were evaluated following the European Medicines Agency "Guidelines on the investigation of Bioequivalence". The phase I, multicenter, randomized, open-label, crossover, single-dose bioequivalence study enrolled 36 adult patients with glioblastoma multiforme or lower-grade glioma. Each patient received 200 mg/m2 Ped-TMZ suspension and TMZ capsules (Temodal®) on 2 consecutive days, with the order being randomly assigned. Fourteen blood samples were collected up to 10 h post-dosing. Bioequivalence was assessed by comparing the 90% confidence interval for the ratio of the geometric means of maximum TMZ plasma concentration (Cmax) and the area under the curve (AUCt). Other endpoints included further pharmacokinetic parameters and safety. RESULTS: Both formulations exhibited a fast in vitro dissolution profile with more than 85% of TMZ dissolved within 15 min. For the bioequivalence study, thirty patients completed the trial as per the protocol. The ratio of Ped-TMZ/TMZ capsule geometric means (90% CI) for AUCt and Cmax were 97.18% (95.05-99.35%) and 107.62% (98.07-118.09%), respectively, i.e., within the 80-125% bioequivalence limits. No buccal toxicity was associated with Ped-TMZ liquid formulation. CONCLUSIONS: This study showed that Ped-TMZ oral suspension and TMZ oral capsule treatment are immediate release and bioequivalent medicines. There were also no unexpected safety signals or local toxicity (funded by ORPHELIA Pharma; ClinicalTrials.gov number, NCT04467346).

Dispensing Oral Temozolomide in Children: Precision and Stability of a Novel and Ready to Use Liquid Formulation in Comparison with Capsule Derived Mixtures.

Temozolomide (TMZ) is part of the therapeutic armamentarium used in managing pediatric cancers; however, available oral forms (capsules) are not adapted for use in children. Our aim was to assess the dose accuracy and stability of TMZ using capsule contents mixed with food compared with a novel, ready-to-use liquid formulation specifically developed for children (Ped-TMZ, brand name KIZFIZO). Dose accuracy and TMZ stability testing were performed with TMZ capsule contents (90 mg) mixed with food vehicles (apple juice, apple sauce, cream, milk, and mashed potatoes) and compared to an equivalent dose of Ped-TMZ. Acceptance criteria were predefined for TMZ (95.0-105.0%) and its degradation product amino-imidazole-carboxamide (AIC; <1%) content. The delivered dose was significantly higher using Ped-TMZ (96.6 ± 1.2%) and within the predefined criteria for TMZ content, whereas it was systematically under the lower specifications of 95% using capsule-derived preparations with apple juice (91.0 ± 1.5%) and apple sauce (91.6 ± 1.4%), respectively (p < 0.0001). In chemical stability tests, the four food vehicles (apple sauce, cream, milk, mashed potatoes) had a significant effect on TMZ stability (p = 0.0042), and the AIC significantly increased with time in three of the four vehicles (p < 0.0001). Only 1/72 of preparations from capsules met the predefined acceptance criteria, whereas Ped-TMZ showed no TMZ loss, and the AIC remained within specifications. In conclusion, mixing TMZ capsule content with food may result in significant underexposure, possibly even greater in routine practice, as complete food intake by the child is unlikely.

Development of a Hospital Compounded, Taste-Masked, Temozolomide Oral Suspension and 5-Year Real-Life Experience in Treating Paediatric Patients.

The development of oral pediatric forms by pharmaceutical companies is still insufficient. In fact, many drugs used in paediatric oncology, such as temozolomide, are not labeled and adapted for paediatric use. Temozolomide (TMZ) is an alkylating agent used as the standard of care for many adult and pediatric brain tumours, such as neuroblastoma, glioblastoma and medulloblastoma. The present study was carried out to propose a suitable and palatable formulation of the oral liquid preparation of TMZ. The suspension is composed of TMZ suspended in SyrSpend SF pH 4, as well as TMZ crystallization stabilizing agents and sweetening agents. To reach this formulation, several taste-masking agents were evaluated. Here, we describe the method of preparation of the formation as well as the monocentric population treated with the formulation over a 5-year period. A 20 mg/mL TMZ suspension was developed. TMZ suspension is stable for 6 weeks, stored between 2 and 8 degrees, protected from light, and compatible with nasogastric tubes. Thirty-eight patients participated in the palatability study and choose cola flavour, and 104 patients were treated in Gustave Roussy with the developed suspension; no unexpected event was reported. To conclude, we propose here a new TMZ liquid formulation which is stable for at least 6 weeks and well-tolerated with extensive feedback.

The Emerging Role of the IL-17B/IL-17RB Pathway in Cancer.

Among inflammatory mediators, a growing body of evidence emphasizes the contribution of the interleukin 17 (IL-17) cytokine family in malignant diseases. Besides IL-17A, the prototypic member of the IL-17 family, several experimental findings strongly support the role of the IL-17B/IL-17 receptor B (IL-17RB) pathway in tumorigenesis and resistance to anticancer therapies. In mouse models, IL-17B signaling through IL-17RB directly promotes cancer cell survival, proliferation, and migration, and induces resistance to conventional chemotherapeutic agents. Importantly, recent work by our and other laboratories showed that IL-17B signaling dramatically alters the tumor microenvironment by promoting chemokine and cytokine secretion which foster tumor progression. Moreover, the finding that elevated IL-17B is associated with poor prognosis in patients with pancreatic, gastric, lung, and breast cancer strengthens the results obtained in pre-clinical studies and highlights its clinical relevance. Here, we review the current understanding on the IL-17B/IL-17RB expression patterns and biological activities in cancer and highlight issues that remain to be addressed to better characterize IL-17B and its receptor as potential targets for enhancing the effectiveness of the existing cancer therapies.

Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies.

Immune checkpoint inhibitors have revolutionized cancer treatment. However, many cancers are resistant to ICIs, and the targeting of additional inhibitory signals is crucial for limiting tumor evasion. The production of adenosine via the sequential activity of CD39 and CD73 ectoenzymes participates to the generation of an immunosuppressive tumor microenvironment. In order to disrupt the adenosine pathway, we generated two antibodies, IPH5201 and IPH5301, targeting human membrane-associated and soluble forms of CD39 and CD73, respectively, and efficiently blocking the hydrolysis of immunogenic ATP into immunosuppressive adenosine. These antibodies promoted antitumor immunity by stimulating dendritic cells and macrophages and by restoring the activation of T cells isolated from cancer patients. In a human CD39 knockin mouse preclinical model, IPH5201 increased the anti-tumor activity of the ATP-inducing chemotherapeutic drug oxaliplatin. These results support the use of anti-CD39 and anti-CD73 monoclonal antibodies and their combination with immune checkpoint inhibitors and chemotherapies in cancer.

The IL-17B-IL-17 receptor B pathway promotes resistance to paclitaxel in breast tumors through activation of the ERK1/2 pathway.

Interleukin 17B (IL-17B) is a pro-inflammatory cytokine that belongs to the IL-17 cytokines family and binds to IL-17 receptor B (IL-17RB). Here we found that high expression of IL-17B and IL-17RB is associated with poor prognosis in patients with breast cancer and that IL-17B expression upregulation is specifically associated with poorer survival in patients with basal-like breast cancer. We thus focused on IL-17B role in breast cancer by using luminal and triple negative (TN)/basal-like tumor cell lines. We found that IL-17B induces resistance to conventional chemotherapeutic agents. In vivo, IL-17B induced resistance to paclitaxel and treatment with an anti-IL-17RB neutralizing antibody completely restored breast tumor chemosensitivity, leading to tumor shrinkage. We next focused on the signaling pathways activated in human breast cancer cell lines upon incubation with IL-17B. We observed that IL-17B induces ERK1/2 pathway activation, leading to upregulation of anti-apoptotic proteins of the BCL-2 family. IL-17B-induced chemoresistance was completely abolished by incubation with PD98059, an inhibitor of the MAPK/ERK pathway, indicating that the ERK pathway plays a crucial role. Altogether our results emphasize the role of the IL-17B/IL-17RB signaling pathway in breast tumors and identify IL-17B and its receptor as attractive therapeutic targets for potentiating breast cancer chemotherapy.

IL-17E synergizes with EGF and confers in vitro resistance to EGFR-targeted therapies in TNBC cells.

Estrogen receptor-, progesterone receptor- and HER2-negative breast cancers, also known as triple-negative breast cancers (TNBCs), have poor prognoses and are refractory to current therapeutic agents, including epidermal growth factor receptor (EGFR) inhibitors. Resistance to anti-EGFR therapeutic agents is often associated with sustained kinase phosphorylation, which promotes EGFR activation and translocation to the nucleus and prevents these agents from acting on their targets. The mechanisms underlying this resistance have not been fully elucidated. In addition, the IL-17E receptor is overexpressed in TNBC tumors and is associated with a poor prognosis. We have previously reported that IL-17E promotes TNBC resistance to anti-mitotic therapies. Here, we investigated whether IL-17E promotes TNBC resistance to anti-EGFR therapeutic agents by exploring the link between the IL-17E/IL-17E receptor axis and EGF signaling. We found that IL-17E, similarly to EGF, activates the EGFR in TNBC cells that are resistant to EGFR inhibitors. It also activates the PYK-2, Src and STAT3 kinases, which are essential for EGFR activation and nuclear translocation. IL-17E binds its specific receptor, IL-17RA/IL17RB, on these TNBC cells and synergizes with the EGF signaling pathway, thereby inducing Src-dependent EGFR transactivation and pSTAT3 and pEGFR translocation to the nucleus. Collectively, our data indicate that the IL-17E/IL-17E receptor axis may underlie TNBC resistance to EGFR inhibitors and suggest that inhibiting IL-17E or its receptor in combination with EGFR inhibitor administration may improve TNBC management.

Regulatory T Cells from Colon Cancer Patients Inhibit Effector T-cell Migration through an Adenosine-Dependent Mechanism.

T cell-mediated immunity is a major component of antitumor immunity. In order to be efficient, effector T cells must leave the circulation and enter into the tumor tissue. Regulatory T cells (Treg) from gastric cancer patients, but not from healthy volunteers, potently inhibit migration of conventional T cells through activated endothelium. In this study, we compared T cells from colon cancer patients and healthy donors to determine the mechanisms used by Tregs from cancer patients to inhibit conventional T-cell migration. Our results showed that circulating Tregs from cancer patients expressed high levels of CD39, an ectoenzyme mediating hydrolysis of ATP to AMP, as a rate-determining first step in the generation of immunosuppressive adenosine. Tumor-associated Tregs expressed even more CD39, and we therefore examined the importance of adenosine in Treg-mediated inhibition of T-cell transendothelial migration in vitro. Exogenous adenosine significantly reduced migration of conventional T cells from healthy volunteers, and blocking either adenosine receptors or CD39 enzymatic activity during transmigration restored the ability of conventional T cells from cancer patients to migrate. Adenosine did not directly affect T cells or endothelial cells, but reduced the ability of monocytes to activate the endothelium. Taken together, our results indicate that Treg-derived adenosine acts on monocytes and contributes to reduced transendothelial migration of effector T cells into tumors. This effect of Tregs is specific for cancer patients, and our results indicate that Tregs may affect not only T-cell effector functions but also their migration into tumors.

CD39: A complementary target to immune checkpoints to counteract tumor-mediated immunosuppression.

We report that CD39-expressing-melanoma cells inhibited both T-cell proliferation and the generation of cytotoxic effectors in an adenosine-dependent manner, and that treatment with a CD39-blocking antibody alleviated tumor-mediated immunosuppression. Thus, blocking CD39 ectonucleotidase may represent a novel immunotherapeutic strategy to restore antitumor immunity.

IL-17A and its homologs IL-25/IL-17E recruit the c-RAF/S6 kinase pathway and the generation of pro-oncogenic LMW-E in breast cancer cells.

Pro-inflammatory IL-17 cytokines were initially described for their pathogenic role in chronic inflammatory diseases and subsequent accumulating evidence indicated their involvement in carcinogenesis. In the present study we report that IL-17A and IL-17E receptors subunits mRNA expressions are upregulated in breast cancers versus normal samples. IL-17E, which is undetectable in most normal breast tissues tested, seems more expressed in some tumors. Investigation of the molecular signaling following stimulation of human breast cancer cell lines with IL-17A and IL-17E showed that both cytokines induced the phosphorylation of c-RAF, ERK1/2 and p70 S6 Kinase were involved in the proliferation and survival of tumor cells. Accordingly, IL-17A and IL-17E promoted resistance to Docetaxel and failed to induce apoptosis as previously reported for IL-17E. Interestingly, we also revealed that both cytokines induced the generation of tumorogenic low molecular weight forms of cyclin E (LMW-E), which high levels correlated strongly with a poor survival in breast cancer patients. These results show for the first time some of the molecular pathways activated by IL-17A and IL-17E that may participate to their pro-oncogenic activity in breast cancers.

Inhibition of CD39 enzymatic function at the surface of tumor cells alleviates their immunosuppressive activity.

The ectonucleotidases CD39 and CD73 hydrolyze extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to generate adenosine, which binds to adenosine receptors and inhibits T-cell and natural killer (NK)-cell responses, thereby suppressing the immune system. The generation of adenosine via the CD39/CD73 pathway is recognized as a major mechanism of regulatory T cell (Treg) immunosuppressive function. The number of CD39⁺ Tregs is increased in some human cancers, and the importance of CD39⁺ Tregs in promoting tumor growth and metastasis has been demonstrated using several in vivo models. Here, we addressed whether CD39 is expressed by tumor cells and whether CD39⁺ tumor cells mediate immunosuppression via the adenosine pathway. Immunohistochemical staining of normal and tumor tissues revealed that CD39 expression is significantly higher in several types of human cancer than in normal tissues. In cancer specimens, CD39 is expressed by infiltrating lymphocytes, the tumor stroma, and tumor cells. Furthermore, the expression of CD39 at the cell surface of tumor cells was directly demonstrated via flow cytometry of human cancer cell lines. CD39 in cancer cells displays ATPase activity and, together with CD73, generates adenosine. CD39⁺CD73⁺ cancer cells inhibited the proliferation of CD4 and CD8 T cells and the generation of cytotoxic effector CD8 T cells (CTL) in a CD39- and adenosine-dependent manner. Treatment with a CD39 inhibitor or blocking antibody alleviated the tumor-induced inhibition of CD4 and CD8 T-cell proliferation and increased CTL- and NK cell-mediated cytotoxicity. In conclusion, interfering with the CD39-adenosine pathway may represent a novel immunotherapeutic strategy for inhibiting tumor cell-mediated immunosuppression.

Lymphocyte-derived interleukin-17A adds another brick in the wall of inflammation-induced breast carcinogenesis.

We have previously reported that a subset of breast tumors are infiltrated with IL-17A-producing tumor-associated lymphocytes and that IL-17A cytokine is principally associated with estrogen receptor negative (ER-) and triple negative, basal-like tumors. We established that IL-17A producing lymphocytes induced cancer cell proliferation, chemoresistance, and invasion, indicating that IL-17A is a potential therapeutic target for breast malignancies.

IL-17A is produced by breast cancer TILs and promotes chemoresistance and proliferation through ERK1/2.

The proinflammatory cytokine Interleukin 17A (hereafter named IL-17A) or IL-17A producing cells are elevated in breast tumors environment and correlate with poor prognosis. Increased IL-17A is associated with ER(-) or triple negative tumors and reduced Disease Free Survival. However, the pathophysiological role of IL-17A in breast cancer remains unclear although several studies suggested its involvement in cancer cell dissemination. Here we demonstrated that a subset of breast tumors is infiltrated with IL-17A-producing cells. Increased IL-17A seems mainly associated to ER(-) and triple negative/basal-like tumors. Isolation of tumor infiltrating T lymphocytes (TILs) from breast cancer biopsies revealed that these cells secreted significant amounts of IL-17A. We further established that recombinant IL-17A recruits the MAPK pathway by upregulating phosphorylated ERK1/2 in human breast cancer cell lines thereby promoting proliferation and resistance to conventional chemotherapeutic agents such as docetaxel. We also confirmed here that recombinant IL-17A stimulates migration and invasion of breast cancer cells as previously reported. Importantly, TILs also induced tumor cell proliferation, chemoresistance and migration and treatment with IL-17A-neutralizing antibodies abrogated these effects. Altogether these results demonstrated the pathophysiological role of IL-17A-producing cell infiltrate in a subset of breast cancers. Therefore, IL-17A appears as potential therapeutic target for breast cancer.

CD39 is involved in mediating suppression by Mycobacterium bovis BCG-activated human CD8(+) CD39(+) regulatory T cells.

Regulatory T (Treg) cells can balance normal tissue homeostasis by limiting inflammatory tissue damage, e.g. during pathogen infection, but on the other hand can also limit protective immunity induced during natural infection or following vaccination. Because most studies have focused on the role of CD4(+) Treg cells, relatively little is known about the phenotype and function of CD8(+) Treg cells, particularly in infectious diseases. Here, we describe for the first time the expression of CD39 (E-NTPDase1) on Mycobacterium-activated human CD8(+) T cells. These CD8(+) CD39(+) T cells significantly co-expressed the Treg markers CD25, Foxp3, lymphocyte activation gene-3 (LAG-3), and CC chemokine ligand 4 (CCL4), and suppressed the proliferative response of antigen-specific CD4(+) T helper-1 (Th1) cells. Pharmacological or antibody mediated blocking of CD39 function resulted in partial reversal of suppression. These data identify CD39 as a novel marker of human regulatory CD8(+) T cells and indicate that CD39 is functionally involved in suppression by CD8(+) Treg cells.

EMT in carcinoma progression and dissemination: facts, unanswered questions, and clinical considerations.

Over the past decade, much effort has been made to understand how cancers metastasize. In deciphering the metastatic process, a vast amount of work has focused on the role of the epithelial to mesenchymal transition (EMT), which, in experimental models, confers tumor cells with invasive and metastatic abilities, resistance to therapies, as well as cancer stem cell phenotype-properties that have a major impact on cancer prognosis. Searching "EMT and cancer" in PubMed retrieves thousands of original research articles, yet, we haven't answered the most basic question in the field: has EMT any relevance in human tumors?

The SNAIL family member SCRATCH1 is not expressed in human tumors.

The SNAIL and SLUG transcription factors play important roles in embryogenesis owing to their anti-apoptotic properties and their ability to promote morphogenetic changes by inducing epithelial-mesenchymal transitions (EMT). These characteristics provide many of the proteins in these families with oncogenic and pro-metastatic capabilities when reactivated in cancers. The SCRATCH subgroup of the SNAIL superfamily, including SCRATCH1 and SCRATCH2, display distinct embryonic functions and diverge early in evolution. Despite the described overexpression of SCRT1 (encoding for SCRATCH1) in a small subset of human lung cancers, there is little data supporting a role of SCRATCH proteins in tumorigenesis. To further explore this possibility, we assessed SNAI1 (SNAIL), SNAI2 (SLUG) and SCRT1 (SCRATCH1) expression in a wide panel of human and murine tumors encompassing 151 primary tumors and 6 different cancer types, including melanomas and multiple different carcinomas. Whereas SNAI1 and SNAI2 are widely expressed in human and murine tumors, our results reveal that SCRT1 transcripts are undetectable in nearly all of the examined tumors suggesting that SCRATCH1 plays a minor role, if any, in tumorigenesis. Our data therefore suggest that oncogenic properties are not shared by all SNAIL superfamily members but instead are specifically allotted to the SNAIL subgroup supporting the conclusions that SNAIL and SCRATCH subgroups are functionally divergent and strengthening the hypothesis that the oncogenic potential of SNAIL and SLUG proteins relies on the hijacking of their embryonic functions.

Interleukin 17 acts in synergy with B cell-activating factor to influence B cell biology and the pathophysiology of systemic lupus erythematosus.

Studies have suggested involvement of interleukin 17 (IL-17) in autoimmune diseases, although its effect on B cell biology has not been clearly established. Here we demonstrate that IL-17 alone or in combination with B cell-activating factor controlled the survival and proliferation of human B cells and their differentiation into immunoglobulin-secreting cells. This effect was mediated mainly through the nuclear factor-kappaB-regulated transcription factor Twist-1. In support of the relevance of our observations and the potential involvement of IL-17 in B cell biology, we found that the serum of patients with systemic lupus erythematosus had higher concentrations of IL-17 than did the serum of healthy people and that IL-17 abundance correlated with the disease severity of systemic lupus erythematosus.

Early origin of cancer metastases: dissemination and evolution of premalignant cells.

Metastasis is the main cause of death by cancer. Hence, establishing predictive markers constitutes a major clinical objective. The capacity for a tumor cell to migrate and survive from a primary tumor is often described as the ultimate step of Darwinian selection. These metastatic cells are believed to emerge from a subpopulation of cells present in a primary tumor. In line with this hypothesis, various gene "signatures" associated with poor prognosis and/or with tumors displaying high metastatic potential have been promoted. However, over the last few years, a growing body of evidence supports the idea that metastatic cells disseminate early from the primordial tumor and evolve independently of it. Herein, we propose to review to the data favoring this alternative model and discuss the interplay between metastatic mechanisms and failsafe mechanism pathways.

Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence.

Twist1 and Twist2 are major regulators of embryogenesis. Twist1 has been shown to favor the metastatic dissemination of cancer cells through its ability to induce an epithelial-mesenchymal transition (EMT). Here, we show that a large fraction of human cancers overexpress Twist1 and/or Twist2. Both proteins override oncogene-induced premature senescence by abrogating key regulators of the p53- and Rb-dependent pathways. Twist1 and Twist2 cooperate with Ras to transform mouse embryonic fibroblasts. Interestingly, in epithelial cells, the oncogenic cooperation between Twist proteins and activated mitogenic oncoproteins, such as Ras or ErbB2, leads to complete EMT. These findings suggest an unanticipated direct link between early escape from failsafe programs and the acquisition of invasive features by cancer cells.